49 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Satoshi Nomakuchi1 *, Junichi Sakagami2 1 Tropical Crop Science, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-8580, Japan ²Tropical Crop Science, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-8580, Japan *Correspondence to: Tropical Crop Science, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-8580, Japan. E-mail: [email protected] ABSTRACT: Tanegashima Island is an inhabited island in Kagoshima Prefecture, where sugarcane is the main industry, and is the northern limit of the cane sugar industry in Japan. Predicting sugarcane yield in the target area is important for increasing sugarcane income in unstable environmental conditions. This study aimed to improve the accuracy of yield prediction by creating a model using climate data related to yield for the past 18 years. The sugarcane growing season is from February to December. We divided the climate data into 3 periods, early growing season from February to April, mid growing season from May to July, and late growing season from August to October, and compared. Multiple regression analysis was used to analyze the relationship between yield, number of stems, and weather. The results, there was a correlation between yield and temperature in the late growing season, while there was no correlation between yield and temperature in the early and mid-growing seasons. The number of productive tillers, which determines yield, was correlated with weather factors in the mid-growing season, but not in the early and late growing season. This suggests that the number of maximum tillers is not a factor in determining yield, the yield is related to the number of productive tillers which is determined in the mid-growing stages. A multiple regression model was created by yield and climate factors for yield prediction. The results of the p-value and estimation were good. Generally, it was believed that a much number of maximum tillers would increase the final yield in the region. But these results suggest that the cultivation system can be improved to limit the number of maximum tillers and to use fertilizer more effectively. Keyword: maximum number of tillers, productive tillers, temperature, growing season, weather factor. O-011 Analysis of Relationship between climate Factors and Sugarcane yield in Tanegashima Island, Kagoshima, based on the farmer’s yielding data.
50 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Jade Benois* École Supérieure d'Agricultures, Angers, 49000, France *Correspondence to: Fairagora Asia 213/10 Asoke Tower Khlong Toei Nuea, Watthana, Bangkok 10110 [email protected] ABSTRACT: Sugarcane production is a crucialsectorinThailand, contributing significantly to the country's economy. However, it is also associated with high greenhouse gas (GHG) emissions, chemical fertilizer use being the main source. To tackle this issue, Fairagora Asia and Nestlé are carrying out a Climate Change Project that aims at reducing the carbon footprint of sugarcane production in Thailand. The results of this study will help to better inform the Climate Change Project, as we aim to identify innovative and sustainable farming practices that can reduce the use of chemical fertilizers by sugarcane producers in Thailand and mitigate GHG emissions. In this presentation, we focus on reviewing sustainable farming practices that are already used in Thailand, such as organic fertilizers, biofertilizers, and varietal selection, and proposes more innovative practices that are not commonly used in the country, such as optimization of chemical spraying, using drones, choosing the right type of fertilizer according to soil type, using frass fertilizer, doing more soil analyses, intercropping or rotating with N-fixing species. The material and methods used in this study will include bibliographic research, stakeholder interviews (researchers, farmers, and industrials), and GHG emissions calculations. We will use Fairagora's CarbonCaneModel, a sugarcane GHG emissions calculator, to estimate GHG emissions reductions from these innovative practices. The interviews, on another hand, will be an important support to gain on-field knowledge and to understand the agro-economic feasibility of each alternative practice in Thai context. Overall, our report will provide recommendations for stakeholders on various sustainable ways to reduce the use of chemical fertilizers in Thai sugarcane production. Keyword: Sugarcane, Thailand, Chemical fertilizers, GHG emissions, Sustainable practices O-016 Innovative sustainable farming practices to reduce chemical fertilizer use in Thai sugarcane production and mitigate greenhouse gas emissions
51 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Khemmanat Thanakronpaisan¹, Ramida Krumsri², Thanatsan Poonpaiboonpipat1 * ¹Department of Agricultural Science, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand, ²No affiliation *Correspondence to: Department of Agricultural Science, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand. Email: [email protected] ABSTRACT: The inhibitory potential of allelopathic plants currently receives much attention to apply them as a tool for weed management in sustainable agriculture. Sugarcane leaves are an agricultural waste product that has been reported to have allelopathic potential. Until now, the optimal organic solvent for sugarcane leaf extraction to yield high-quality and allelopathic active compounds for developing natural herbicides has not yet been studied. Therefore, the present study was to determine the optimal organic solvent system and fractionation procedure to enhance sugarcane leaf extracts' quantity and allelopathic efficiency. Sugarcane leaves were extracted using different ethanol: water solvent ratios (00:100, 25:75, 50:50, 75:25, and 100:00 (v/v)). Their allelopathic effects were assayed for seed germination and seedling growth in two major weeds, Echinochloa crus-galli, and Amaranthus viridis. Results showed that extract concentrations, solvent ratios, and their interactions significantly inhibited all growth parameters in A. viridis. In contrast, no significant inhibitory effects were observed on E. crus-galli. The quantity of crude extracts was found to be dependent on solvent proportion. Among them, a mixture of ethanol and water at a ratio of 100:00 (v/v) had the most inhibition on A. viridis and produced the highest quantity of crude extracts. Consequently, crude ethanol: water (00:100 (v/v)) was conducted to separate the active fraction by acid-base solvent partitioning into four fractions: the crude ethanol extract fraction (OR), an acidic fraction (AE), a neutral fraction (NE), and an aqueous fraction (AQ). Fraction NE exerted the highest inhibitory effects with complete (100%) inhibition of A. viridis at all concentrations, followed by the fractions of OR (IC50 = 3008 to 3976 ppm), NE (IC50 =1821 to 6927 ppm), and AQ (IC50 =8763 to 10089 ppm), respectively. These findings suggested that the fraction of NE separated from ethanol sugarcane leaf extracts might contain the main potential allelopathic substances. It could be used as a candidate source for isolating their active substances for future utilization as natural herbicides. Keywords: Allelopathy, Sugarcane leaves, Optimal extraction solvents, Natural herbicides O-020 Allelopathic effects of sugarcane leaves: optimal extraction solvent and partial separation of their potent allelopathic active fractions
52 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Kenta Watanabe1 *, Eizo Taira¹, Muneshi Mitsuoka¹, Masami Ueno², and Yoshinobu Kawamitsu¹ 1 Faculty of Agriculture, University of the Ryukyus, Nishihara, 9030213, Japan ²NPO Subtropical Biomass Research Center *Correspondence to: Faculty of Agriculture, University of the Ryukyus, Nishihara, 9030213, Japan. E-mail: [email protected] ABSTRACT: Sugarcane is one of the most important agricultural products in the southwest Japan with an economic impact greater than that of any other crop. In the last decade, the proportion of machinery use in sugarcane cultivation has been dramatically increasing due to the decreasing number and aging of sugarcane growers. Moreover, the labor shortage has led to extensive and improper field managements, resulting in lower yield and quality and unstable sugarcane production that is easily influenced by the climate of the year. In short, development of machinery operation systems and improvement in yield and quality are major concerns for the establishment of stable sugarcane production in the country. To solve this problem, we launched a smart farming project called “UFSMA” in 2019 and have developed and introduced a number of technologies such as (1) automatic steering of agricultural machinery using global navigation satellite systems (GNSS), (2) Micro weather stations and its data streaming systems, (3) water-saving remote irrigation systems based on micro weather data, (4) sugarcane growth monitoring using unmanned aerial vehicle (UAV), (5) sugarcane quality prediction using mobile NIRS equipment and UAV, (6) pest, disease and weed control using UAV, (7) geographic information system (GIS)-based farming support systems, and (8) big data analysis. The project is still ongoing, and our goals by utilizing these technologies efficiently are as follows: establishment of highly accurate and labor-saving cultivation systems by automatic steering of agricultural machinery, increase in sugarcane yield and quality by precise irrigation systems based on sugarcane growth and environment data, integration of sugarcane growth, environment, and economics data into the farming support systems and its efficient use, and enhancement of functions as a production model by using big data information. Besides, challenges to achieve the goals are also discussed. Keyword: GNSS, micro weather station, smart irrigation, UAV, GIS-based farming support systems O-022 Smart farming for sugarcane cultivation in Japan: current status and perspectives
53 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE S.N. Singh¹, T. K. Srivastava¹, A. K. Sah¹, C. Gupta¹, V. K. Singh¹ and J. R. Patnaik² 1 ICAR-Indian Institute of Sugarcane Research, Lucknow-226002, India ²Sugarcane Research Station (OUAT), Nayagarh-752070 (Odisha), India ABSTRACT: A technology for saving of precious seed cane material and rapid multiplication of newly released varieties of sugarcane was developed by ICAR-Indian Institute of Sugarcane Research, Lucknow. This technology has successfully addressed the concerns of seed cane production of promising and newly released varieties of sugarcane besides reducing the cost of cultivation. To introduce and validate the technology at farmers’ fields, an on-farm trial at the different sugar mill zones (east, central and west) of Uttar Pradesh was conducted in two farmers’ fields each at two villages separately totaling to 12 numbers of farmers in all for every three cropping seasons of 2016-17, 2017-18 and 2018-19. Experimental results indicated that the cane yield obtained in different farmers’fields of all the three sugar factory zones exhibited almost the same trend with that of germination %, tillers and millable cane numbers. On an average, the cane yield of 95.11 t/ha was obtained under cane node planting technique from all the three sugar factory zones, and it was higher by 8.88% than that of conventional method of planting (86.66 /ha). The components of cost of cultivation clearly indicate that seed cane and preparation account for 22% of the total cost of cultivation. This cost was significantly reduced with the adoption of cane node technology using only 17 q/ha of seed cane quantity as against 70 q/ha under conventional 3-bud setts of sugarcane planting. The results indicated that cane node technology raised sugarcane multiplied seed cane by 34 times as against 10.3 times under conventional sett method of planting. The benefit cost ratio was higher in case of cane node method of sugarcane planting (1.90) as against 1.47 under conventional 3 bud setts planting. The technology of sugarcane planting by cane node method is becoming popular among sugarcane farmers in different zones of the state since it gives not only good cane yield but also saves precious seed cane planting material and thereby reduces cost of cultivation. Sugarcane farmers are also preferring this technology and using it for rapid multiplication of newly released varieties of sugarcane. O-027 Assessing cane node planting technique for saving of precious seed cane material and rapid multiplication of seed cane with reduced cost at farmers’ fields in Indian subtropics
54 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Shotaro Ando1 *, Yoshifumi Terajima¹, and Youichi Kobori² ¹Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences, Ishigaki, 9070002, Japan ²Japan International Research Center for Agricultural Sciences, Tsukuba, 3058686, Japan Correspondence to: Tropical Agriculture Front, Japan International Research Center for Agricultural Sciences, 1091-1 Kawarabaru, Maezato, Ishigaki, 9070002, Japan. [email protected] ABSTRACT: Japan International Research Center for Agricultural Sciences (JIRCAS) conducts collaborative research projects related to sugarcane in Thailand together with Thai organizations, such as Khon Kaen Field Crop Research Center, Department of Agriculture and Khon Kaen University for more than 20 years. At first, we collected genetic resources related to sugarcane from all over Thailand. Wild sugarcane (Saccharum spontaneum) and Erianthus were conserved and evaluated their morphological, agronomic, and genetic characteristics. Recently we developed database of Erianthus germplasm on the home page of JIRCAS (https://www.jircas.go.jp/ja/database/erianthus). We developed new cultivar, TPJ04-768, using an interspecific crossing between sugarcane and S. spontaneum. The sugar yield of this cultivar was comparable to that of the conventional cultivar KK3 although the sugar content was slightly lower. Moreover, the production of fiber (bagasse) in this cultivar is approximately 1.5 times higher than that in KK3 in Northeast Thailand. Thus, TPJ04-768 is a suitable raw material for biofuel and other biomass applications. TPJ04-768 is also suitable for multiple ratoon cultivation based on its decreased yield reductions in ratoon cropping. Erianthus, sugarcane-related germplasm, has potential as breeding material for sugarcane improvement because of its desirable traits such as high biomass productivity, vigorous ratooning, drought tolerance, and disease resistance. New breeding materials were developed through intergeneric crossing between sugarcane and Erianthus for further improvement of sugarcane productivity and adaptability to adverse environments. To control sugarcane white leaf disease, we developed a system for propagation of healthy seed cane including field management techniques for propagation, distribution methodsfor the production, proceduresto detect pathogens using LAMPmethod, etc. Manual of thissystem was published on the home page of Office of Cane and Sugar Board (https://www.ocsb.go.th/wp-content/ uploads/2023/03/12686-3399.pdf). Verification test of propagation of healthy seed cane until the third generation demonstrated sufficiently low rate of diseased plants. Keyword: Erianthus, healthy seed cane, Saccharum spontaneum, sugarcane white leaf disease O-028 Review of collaborative research projects of JIRCAS on sugarcane in Thailand
55 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Jariya Roddee¹,4, Suchawadee Klangjoho1 , Napatson Chansawang1 , Jureeemat Wangkeeree² and Yupa Hanboonsong³ 1 School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, Nakhon Ratchasima, Thailand 2 Department of Agricultural Technology, Faculty of Science and Technology, Thammasat University Rangsit Centre, Khlong Nueng, Klong Luang, Pathum Thani, Thailand ³Department of Entomology and Plant Pathology, Faculty of Agriculture, Khon Kaen University, Nai Muang, Muang, Khon Kaen, Thailand 4 Innovation of Quality Enhancement of Agricultural Products for Agro-Industry-Research Center, Suranaree University of Technology, Suranaree, Muang, Nakhon Ratchasima, Thailand *Correspondence to: School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, Nakhon Ratchasima, Thailand. E-mail [email protected] ABSTRACT: The Yamatotettix flavovittatus Matsumara is a leafhopper vector of sugarcane white leaf (SCWL) phytoplasma that causessugarcane chlorosissymptoms.The effects ofstylet probing (or penetration) behaviors of Y. flavovittatus on sugarcane and its implication for SCWL phytoplasma transmission are yet to be studied. In this research, we used DC electropenetrography (EPG) to define waveforms produced by an adult and fifth-instar nymphal Y. flavovittatus on sugarcane and scanning electron microscopy. The following six waveforms and associated activities are described: (NP) non-probing, (Y1) stylet probing into epidermal cells, (Y2) stylet probing through mesophyll/parenchyma, (Y3) stylet contact with phloem and likely watery salivation, (Y4) active ingestion of sap from phloem, probably sieve elements, and (Y5) unknown stylet activity in multiple cell types. Study findings reveal that the Y. flavovittatus vector ingests phloem sap more frequently and for longer durations than from any other cell type, supporting that Y. flavovittatus is primarily a phloem feeder. Adult Y. flavovittatus show a longer total probing duration and produces high density of puncture holes on sugarcane leaves. Moreover, stylet probing behaviors revealed that adults typically ingest phloem sap more frequently and for longer durations than do fifth-instar nymphs, enhancing nutrition. Furthermore, we propose that adults are more likely to both acquire (during Yf4) and inoculate (during Yf3) higher amounts of phytoplasma than fifth-instar nymphs. This information on the stylet penetration behavior of leafhopper Y. flavovittatus serves is a basis for advanced studies on the transmission mechanism of SCWL phytoplasma. Keywords: feeding behavior, insect-plant interaction, phytoplasma, stylet penetration, sugarcane white leaf disease. O-029 Stylet Penetration of Yamatotettix flavovittatus Matsumura (Hemiptera: Cicadellidae) on sugarcane by DC electropenetrography and scanning electron microscopy
56 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION Yamatotettix flavovittatus Matsumura (Hemiptera: Cicadellidae) is an insect vector of the sugarcane white leaf (SCWL) disease (Hanboonsong et al., 2002, 2006)—one of the most dangerous diseases and cause of significant sugarcane yield losses in Asian countries, especially in Thailand. The SCWL disease is caused by a phytoplasma pathogen, not yet named, which is an obligate parasite of the host cell and cannot be cultured. The SCWLphytoplasma is transmitted and spread by infected seed canes or one of its leafhopper vectors, Y. flavovittatus or Matsumuratettis hiroglyphicus (Matsumura) or (Hanboonsong et al., 2002, 2006). A reported technique for controlling Y. flavovittatus is using sticky traps to inhibit the dispersal behavior of the insect vector (Thein et al., 2011). Bacterial symbionts have also been identified for possible use in insect population control, including finding the secondary bacterial genus Wolbachia, an intracellular, maternally inherited bacterium. Wolbachia's population dynamics and phylogeny in the Y. flavovittatus leafhopper have been examined (Wangkeeree et al., 2020a, 2021a, 2021b). Wolbachia infection affects the life cycle and reproduction system of the Y. flavovittatus leafhopper (Wangkeeree et al., 2020b). The electropenetrography (EPG), developed over the past 60 years, employs an electronic monitor to measure stylet probing behaviors of insects with piercing-sucking mouthparts, in real-time. EPG has been widely utilized for research on sap-sucking insects, such as studies of insect resistance to insecticides, factors influencing plant resistance to insects, insect vector feeding behavior, and stylet penetration activities linked to pathogen transmission mechanisms. Hemipteran feeding behavior has been intensely and rigorously studied using EPG monitors since the 1960s, when D.L. McLean and M. G. Kinsey developed the first AC (alternating current) electronic monitors (AC EPG) (Mclean and Kinsey, 1964). Monitors were then improved when W. F. Tjallingii employed a DC (direct current) and higher amplitude sensitivity (fixed at input resistor [Ri] 109 Ohms) and renamed the electrical penetration graph (DC EPG) system (Tjallingii, 1978, 1988). Backus and Bennett (1992) developed and tested the newer, AC-DC EPG, which combined theAC and DC monitor design features with tunable settings(Backus and Bennett, 2009; Rangasamy et al., 2015). All EPG systems have resulted in numerous studies on piercing-sucking insects, such as those exploring the effect of stylet probing and ingestion behaviors on rice plant tissue of the Sogatella furcifera (Horváth) and Nilaparvata lugens(Stål) planthoppers(Seo et al., 2009; Kang et al., 2022).Also, the feeding behaviors of M. persicae aphid feeding on Capsicum annuum (pepper) (Jacobson and Kennedy, 2013) were studied, as were Diaphorina citri Kuwayama psyllid (Bonani et al, 2010); pear psyllids (Civolani et al., 2011); Graphocephala atropunctata Signoret sharpshooter leafhopper (Signoret) (Miranda and Fereres, 2009), and the like. Correlations have been studied between waveforms and stylet activities of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Dugravot et al., 2008); the Graminella nigrifrons (Forbes) leafhopper (Jin et al., 2012; Gerstenbrand et al., 2021; Roddee et al., 2021). The study’s results provide basic information on the behaviors of the Y. flavovittatus vector, which can be utilized in further studies on the localization of sugarcane resistance in different varieties and studies on the transmission mechanism of SCWL phytoplasma. The objectives of this study were to (1) study the EPG waveforms and their characteristics produced by adult Y. flavovittatus leafhopper vectors, (2) compare the feeding behavior of adult Y. flavovittatus leafhopper vectors using a scanning electron microscope (SEM). 2. MATERIALS AND METHODS 2.1 Insect and plant materials Y. flavovittatus leafhoppers were developed from insects collected in a sugarcane field in the Nakorn Ratchasima Province, northeast Thailand, using light traps (black light). The insects were reared on disease-free sugarcane plants (Saccharum officinarum) (KK3 variety) grown from tissue culture. The leafhoppers wase mass in caged pots under supplemental light at 27 ± 2 °C Sugarcane fodder plants were changed every 2 weeks to achieve minimum SCWL phytoplasma colonization of vectors. The stylet probing study employed 5–7-day-old adults. One-month-old sugarcane plants obtained from the Eastern Sugar and Cane Public Company Limited, Sa-Kaeo Province were individually transplanted into pots (16 cm diameter, 12 cm high), covered with clear plastic cages (15 cm diameter, 35 cm high), and reared in the greenhouse under natural light at 26 °C–30 °C. The plant was 8 weeks past the second leafing stage at use. 2.2 Electropenetrography methods. Y. flavovittatus leafhoppers adults were removed from the colony and individually starved for 40 min in glass tubes. They were then placed inside an ice box for 3—4 min to make them quiescent and reduce their metabolic rate, whereupon the insects were attached to a gold wire electrode under a microscope at the pronotum right between the wings using tweezers and silver glue. A Giga-8 DC-EPG system (manufactured by EPG Systems, W.F. Tjallingii, Wageningen University, Netherlands) with 1 Giga-ohm (109 Ohms) input resistance (Ri) was employed for EPG recording. Each insect was connected to one of the primary amplifier inputs before being placed on the third or fourth sugarcane leaf from the terminal, secured using parafilm strips (Bemis, USA). The plant electrode for conveying
57 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE substrate voltage was inserted into the soil of the potted sugarcane, and the substrate voltage was adjusted such that the EPG signals could range from −5V to +5V. The plant was placed in a plastic pot, and the insect and amplifiers were placed inside a Faraday cage to cancel out the electrical field and insulate the contents of the cage (one leafhopper per plant). Eight leafhoppers (male and female) were recorded for 8 hours per day. The investigation was designed using a completely randomized design. Stylet probing of Y. flavovittatus was recorded and analyzed employing the STYLET+ software Tjallingii, and Hogen, (1993) to manually distinguish the different waveforms according to their specific patterns. The electrical signals produced by the probing behaviors of the insects were recorded on a computer using the EPG output set at 50X–100X gain. The mean and standard errors, of amplitude and frequency range were calculated based on 42 waveform events. 2.3 Waveform measurement and data analysis Probing behaviors of Y. flavovittatus were calculated by the number of probes produced by leafhoppers and the total duration of the probing (Pr) phase versus the non-probing (NP) phase (Roddee et al., 2017). After measuring the typical waveforms related to Y. flavovittatus probing (see Results), the following variables (mean ± standard error) were calculated, as described in Backus et al. (2007): the total probing duration (TPD; min); total waveform duration (TWD, sec); a number of waveform event per insect; (NWEI, time); waveform duration (WDI; min) per insect. Variable values over an 8-hour access period were statistically compared among adult males, and adult females for waveforms NP, A, B, C, D, and F using the Stylet program with non-parametric analyses. EPG parameters (mean ± standard error) were conducted using the Tukey–Kramer at P < 0.05, as SAS program (Backus v. 2.0 described in Backus et al. (2007). 2.4 Stylet penetration holes of Y. flavovittatus via scanning electron microscopy (SEM) SEM was utilized to observe puncture holes left post-probing by Y. flavovittatus by adult females, and adult males. A total of 20 adults were caged on the third leaf from the leaf shoots of sugarcane. To confirm the probing site by finding the salivary flange, the sheath materials on the adaxial surface of each leaf were stained red with 1% eosin water solution after the insects were done feeding, according to the method by Roddee et al. (2017). After staining, each living leaf was cut into 5×5 mm pieces, fixed with 6% paraformaldehyde overnight, and washed three times in phosphate-buffered saline (PBS). The sugarcane leaf tissues were then dehydrated in an increasing ethanol series, critical-point dried, and coated with gold-palladium. The fixed surfaces of the leaves were then observed under an SEM (SEM™, FEI, Quanta450, Netherlands). 3. RESULTS AND DISCUSSION 3.1 The stylet probing behavior of the leafhopper, Y. flavovittatus using EPG The following six different waveforms were identified for Y. flavovittatus leafhoppers: NP, A, B, C, D, and F. After being placed on the leaf surface, males, and females showed a short period (10–15 seconds) of NP, non-probing. Subsequently, stylets probed into the sugarcane leaf (Pr), generating a series of waveforms interspersed with NP events. During the Pr phase, five different waveforms were identified: A, waveform B, waveform C, waveform D, and waveform F according to the corresponding probing behaviors or activities on the plant tissue. Some probing waveforms observed for Y. flavovittatus show similar characteristics to those observed for M. hiroglyphicus (Roddee et al., 2017, 2019). This is likely because they are both piercing-sucking leafhoppers (family Cicadellidae) that belong to the same subfamily, Deltocephalinae. Many notorious leafhopper vectors are deltocephalines, as are the identified vectors of the SCWL phytoplasma (Hanboonsong, 2002, 2006). Despite this similarity in waveforms between the two species, some of the waveforms (C and D) exhibited more details for Y. flavovittatus than for M. hiroglyphicus (C and D waveforms). Moreover, some of the probing waveforms observed for Y. flavovittatus were also found in other piercing-sucking insect groups, such as other leafhoppers (Lett et al., 2001; Stafford and Walker, 2009) Total waveform duration (TWD) was different among life stages. A quantitative analysis of probing behavior, female Y. flavovittatus performed the most total stylet probing duration (TPD) (56%) than males (54%) (Figure 1). The leafhopper Y. flavovittatus both male and female spent more probe duration on A waveform (Figure 1).
58 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Figure 1. TWD and TPD of male, and female Y. flavovittatus leafhoppers feeding on sugarcane leaves during an 8-hour recording. The number of waveformsA,B,C, D, and F did notsignificantly differ among the male and female Y. flavovittatus. The probe durations per insect of females were significantly different higher males (Table 1). The male leafhopper Y. flavovittatus spent longer in A and D waveforms than the female leafhopper Y. flavovittatus (Table 1). White female leafhopper Y. flavovittatus spent longer in C waveforms than male leafhopper Y. flavovittatus (Table 1). Table 1 Mean SEM electrical penetration graph (EPG) waveforms and parameters measured during 8 h-recording for male and female Yamatotettix flavovittatus leafhopper on Knon Kean 3 sugarcane cultivar. 3.2 Stylet probing of Y. flavovittatus examined through scanning electron microscopy. The stylet probing behaviors of male and female Y. flavovittatus leafhoppers were compared by examining the puncture holes that the insects produced after stylet probing (Figure. 2). Puncture holes produced by male Y. flavovittatus leafhoppers were higher in number (3–4 puncture holes) (Figure 2A) and significantly different in appearance than those produced by female Y. flavovittatus per 100 µm (Figure 2B) of the sugarcane leaf surface. This means that male leafhoppers showed a higher probing frequency than female Y. flavovittatus counterparts. Our results indicated that the WDI of phloem sap ingestion, waveform D) was higher for adult female Y. flavovittatus leafhoppers than for their male counterparts. Waveform duration per insect (WDI) was also significantly greater
59 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE for females. This difference predicts that females will be more effective vectors of SCWL phytoplasma than males and may partially explain the reported gender-based differences in transmission efficiency (Roddee et al., 2019). While the pathway phase is observed more frequently in males than females, gender-related ability to transmit pathogens is also associated with leafhopper vector species. For example, Chuche and Thiéry (2014) reported that the males of the leafhopper Scaphoideus titanus Ball, which is a vector of grapevine flavescence dorée phytoplasma, produced more frequent and longer probes in general in the phloem than females. Our previous study on the female M. hiroglyphicus highlighted that they exhibited more frequent probing and phloem penetration behavior for a longer duration than males; therefore, the females are likely to have a greater probability of acquiring and inoculating the SCWL phytoplasma. (Roddee et al., 2017, 2019). Figure 2. Scanning electron microscopy (SEM) images of stylet punctures produced by Y. flavovittatus on the sugarcane leaf surface. (A and C) the distribution of stylet holes and salivary sheaths of male Y. flavovittatus (B and D) stylet holes left of female Y. flavovittatus 4. CONCLUSIONS In conclusion, this is the first EPG study to investigate the characteristics of stylet probing behaviors of Y. flavovittatus, using light microscopy and SEM. The information on the stylet probing of the Y. flavovittatus leafhopper on sugarcane leaves should serve as a basis for advanced studies on the transmission mechanism of SCWL phytoplasma. The study results can facilitate the development of more effective methods to interfere with this process to reduce disease spread in sugarcane fields, such as breeding sugarcane resistance to insect vectors. We have described the fundamental DC EPG waveforms associated with the stylet pathway and subsequent activities in phloem where insect acquisition and inoculation of the SCWL disease is most likely to occur. Further study should determine the acquisition and inoculation of the SCWL disease and estimate the time thresholds of vector transmission of this disease in sugarcane. 5. ACKNOWLEDGEMENT This work was financially supported by the Office of Permanent Secretary, Ministry of Higher Education, Science, Research, and Innovation (Grant No. RGN 63-116) and Suranaree University of Technology.
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61 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Seo, B. Y., Kwon, Y. H., Jung, J. K. and Kim, G. H. (2009). Electrical penetration graphic waveforms in relation to the actual positions of the stylet tips of Nilaparvata lugens in rice tissue. J. Asia-Pac. Entomol. 12, 89–95. Stafford, C. A. and Walker, G. P. (2009). Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus. Entomologia Experimentalis et Applicata. 130, 113–129 Tjallingii, W. F. (1978). Electronic recording of penetration behavior by aphids. Entomol. Exp. Appl. 24, 721–730. Tjallingii, W. F. (1988). Electrical recording of stylet penetration activities. In: Minks, A. K., Harrewijn, P. (eds.), World Crop Pests, vol. 2b. Aphids, Their Biology, Natural Enemies and Control. Elsevier, Amsterdam, The Netherlands, pp. 95–108. Tjallingii, W. F. and Hogen, E. Th. (1993). Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals). Physiology Entomology.18, 317–328. Thein M. M., Jamjanya, T.; Hanboonsong, Y., 2011. Evaluation of colour traps to monitor insect vectors of sugarcane white leaf phytoplasma. Bulletin of Insectology. 64, 117-118. Wangkeeree, J., Sanit, P., Roddee, J., Hanboonsong, Y., 2021a. Population Dynamics of Wolbachia in the Leafhopper Vector Yamatotettix flavovittatus (Hemiptera: Cicadellidae). Journal of Insect Science. 21(6), 1–8. Wangkeeree J., Sanit, P., Roddee, J., Hanboonsong, Y., 2021b. Phylogeny and Strain Typing of Wolbachia from Yamatotettix flavovittatus Matsumura Leafhoppers. Current Microbiology. 78(4), 1367 – 1376. Wangkeeree J., Tewaruxsa, P., Roddee, J., Hanboonsong, Y., 2020a. Wolbachia (Rickettsiales: Alphaproteobacteria) infection in the leafhopper Vector of sugarcane white leaf disease. Journal of Insect Science. 20(3), 1–8. Wangkeeree J., Suwanchaisri, K., Roddee, J., and Hanboonsong, Y.., 2020b. Effect of Wolbachia infection states on the life history and reproductive traits of the leafhopper Yamatotettix flavovittatus Matsumura. Journal of invertebrate pathology. 177, 107490.
62 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Natthapol Chittamart¹, Pechrada Pinjai¹, Saowanuch Tawornpruek1 * and Supaporn Lertsiri² ¹Department of Soil Science, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand ²Agriculturul Extension and Communication, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand Correspondence to: Department of Soil Science, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand. [email protected] ABSTRACT: Implementing nutrient management technology, including organic residue mulching and nutrient-enhancing microorganisms, significantly boosts sugarcane soil productivity, helps growers effectively manage their soils, and increases yields can support BCG economic policy growth. This research focuses on transferring soil-fertilizer management technology to sugarcane-growing areas in Sa Kaeo Province. Representative sugarcane planting soils with clayey texture, low organic matter, low potassium reserves, and drought risk were selected for demonstration plots. Soil samples were collected after the first sugarcane harvest, and fertilizer rates were determined using the integrated nutrient management (KU model), compared to traditional farmer's soil management (FM model). The demonstration plots of KU-Model were set up in three locations to compare the effects of leaf residue and site-specific nutrient management along with applying phosphate solubilizing bacteria (PSB). Cane yield, soil fertility, and fertilizer cost were assessed and compared to conventional management. The study revealed that the initial soil conditions in the three tested areas had slightly acidic to neutral pH (6.25-7.31), low to moderate organic matter, nitrogen, phosphorus, and potassium levels. Nitrogen, phosphorus, and potassium were identified as the primary limitations. Additionally, the soil exhibited high calcium and magnesium content, potentially reducing sugarcane’s potassium uptake. Comparing the two soil management models, the KU model effectively increased and maintained macronutrient levels in the soil, resulting in more sugarcane shoots and increased yield compared to the FM model. Yield increases ranged from 30-40%, corresponding to a revenue boost of 2,640-3,240 baht per rai at a market price of 1,200 baht per ton. The value-cost ratio (VCR) ranged from 1.2-1.5, indicating that adopting the KU model for fertilizer management proved economically viable. Furthermore, the fertilizer applied in the experimental year served as nutrient reserves for second ratoon cane without requiring additional fertilization. Implementing phosphate-soluble bacteria (PSB) manipulation enhanced phosphorus availability from most soil phosphorus in iron oxide and calcium phosphate forms. The KU model exhibited a higher population of phosphate-soluble bacteria than the FM model, indicating that microbial spraying stimulated their growth and improved phosphorus utilization during the sugarcane growing season. Farmers readily accepted the KU model soil management technology, providing an alternative practice to enhance soil and nutrient management, increase yield, and improve sugarcane quality in subsequent phases of the research. Keyword: soil management, fertilizer management, ratoon cane yield, BCG economy, PSB O-032 Enhancing soil fertility, reducing fertilizer costs, and boosting ratoon cane yields through integrated nutrient management technology in Sa Kaeo province, Thailand
63 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Siriya Kumpiro1,2*, Sopon Uraichuen1 1 Department of Entomology, Faculty of Agriculture at Kamphaeng Saen Campus, Kasetsart University, Nakhon Pathom, 73140, Thailand ²Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, 50200, Thailand *Correspondence to: Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, 50200, Thailand. [email protected] ABSTRACT: Sugarcane shoot and stem borers (SSSBs) are destructive pests that cause significant damage to sugarcane crops. SSSB larvae damage sugarcane plants by boring or tunneling inside their plant stems. While parasitoids are commonly used as natural enemies for SSSB control, their effectiveness may vary in certain seasons or locations. Insect diseases caused by entomopathogenic fungi offer an alternative approach for biological control. This study aimed to survey and collect entomopathogenic fungi associated with SSSBs and evaluate their pathogenicity through laboratory experiments by topical application. A completely randomized design (CRD) was set as the experimental design. Three entomopathogenic fungi belonging to the genera Fusarium, Purpureocillium, and Metarhizium were isolated from Chilo tumidicostalis larvae, a species of SSSB. These isolates were found to infect C. tumidicostalis after 2 days of inoculation. The mortality rates and infestation times varied among the isolates, with M. anisopliae MSS showing the highest mortality rate (87.50±5.00%) and fastest infestation time (LT50 value of 1.003 days). Fusarium sp. FSS and P. lilacinum PSS exhibited lower mortality rates (50.00±8.16% and 40.00±14.14%, respectively) and longer infestation times (LT50 values of 7.77 days and 13.06 days, respectively). These promising entomopathogenic fungi could be further developed as biological agents for the control of SSSBs Keywords: : Entomopathogenic fungi, sugarcane shoot and stem borers, M. anisopliae, Fusarium sp., P. lilacinum O-033 Entomopathogenic Fungi Isolated from Sugarcane Shoot and Stem Borer
64 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION Sugarcane is a vital industrial crop in Thailand, contributing significantly to the country's economy through the production of sugar and renewable energy., it has hence encouraged the production of sugarcane to increase. And Policy Agricultural Thailand reformed in 2013-2017 has to transform the area unsuitable for growing rice to other economic crops such as sugarcane and would to expand the sugarcane planting area from a sugar factory radius of 50 kilometers in each district in all the province. However, sugarcane cultivation is frequently plagued by insect pests, with the shoot and stem borers (SSSBs) being the most damaging. The SSSBs, including Chilo infuscatellus, Chilo sacchariphagus, Chilo tumidicostalis, Sesamia inferens, and Scirpophaga excerptalis, [20]. SSSBs can infest sugarcane at various growth stages, causing hollowing of the stems, withering of shoots, and significant economic losses in terms of quantity and quality. [13]. The infestations of stem borers negatively affected plant growth and sugarcane production. Different sugarcane characters were recorded including height, diameter, weight, brix, pol, purity, fiber and CCS. The data of all characters varied, depending upon sugarcane varieties. The reduction in stalk weight ranged from 16.59-36.53 percent the pol loss ranged from 4.03-33.95 percent. the reduction in CCS ranged from 3.76-40.60 percent [9]. Chemical insecticides have traditionally been used for SSSB control but have negative environmental impacts and can lead to insecticide resistance. [15]. Integrated pest management (IPM) approaches, including the use of parasitic wasps, have been adopted, but their effectiveness can be limited in certain seasons. As a result, the exploration of alternative natural enemies, such as entomopathogenic fungi, is crucial for sustainable SSSB management. They exist in the food chain and can maintain the natural balance in the ecosystem. The infestation of insect fungi is not like BT or NPV that insects must eat. But the fungi attack insects by penetrating mycelium through the body cuticle and growing inside the body of the insect causing the insect to die. There are no reports of fungi that cause insect diseases against SSSBs in Thailand. This study was to collect fungal pathogens of SSSBs. and further, develop microbial control of the sugarcane shoot stem borer. 2. MATERIALS AND METHODS Collection of Entomopathogenic fungi of Sugarcane shoot and stem borers The study involved the collection of SSSBs from six sugarcane plantations in Suphan Buri Province. Selecting and cutting sugarcane that shows signs of infestations were pinholes on leaves, tiny holes on the midribs, and holes on stalks. Keep and transfer in the plastic box were isolated and subjected to morphological identification in the laboratory to be continued. Isolation and Identification of entomopathogenic fungi The surface of dead larvae was sterilized by 2% sodium hypochlorite solution for 3 minutes, rinsed in sterile distilled water, then dried using sterile filter paper. The fungi were isolated directly from dead larvae by taking fruiting body and conidia onto PDA with 0.2 mg/l chloramphenicol and incubated at 25±2°C for 5 days. For pure culture isolation, the mycelial tip of a small fungal colony was cut and transferred to new plates. Pure cultures were also incubated at 25±2°C. The entomopathogenic fungi were isolated from the larvae. The isolated fungi were cultured on Potato Dextrose Agar (PDA) plates and identified based on their morphological characteristics. [8, 10,] Pathogenicity of entomopathogenic fungi against C. tumidicostalis larva in laboratory The larva and pupa of C. tumidicostalis used in this bioassay were collected from sugarcane plantations. They were then placed in plastic boxes with the cane for feed until they pupated and became adults. Then 10 pairs of adults were released into net cages and supplied with 2 months old sugarcane for laying eggs and cultured as larvae for experimentation. To evaluate the pathogenicity of the isolated fungi, laboratory experiments were conducted. Healthy larvae of C. tumidicostalis were used for the bioassays. Each fungal isolate was grown on PDA plates for 14 days at 25°C. The conidia were then harvested and suspended in a solution containing 0.01% Tween 80. Ten fourth instar larvae of pink stem borer were placed in plastic boxes together with freshly chopped cane for food. For each isolate an aqueous conidia suspension was prepared with 0.01 percent of Tween 80 for 1.0 x 108 conidia per milliliters and applied 5 microliters by volume using micropipette on larvae body. A control group of larvae treated with a solution of 0.01% Tween 80 was also included. The treated larvae were kept in a growth chamber at 27℃ and 70 ± 5% RH. Complete Randomized Design (CRD) with 4 replications were set as the experimental design. The mortality of the larvae was recorded daily for a period of 10 days. Dead larvae were surface-sterilized, placed on PDA plates, and observed for fungal growth to confirm the cause of death. The data obtained from the bioassays were analyzed to determine the mortality rates (Mortality data was corrected for the corresponding Control Mortality by the Abbott’s formula [1]) and infestation times of the larvae. The LT50 (time required to cause 50% mortality) values were calculated using probit analysis.[14]
65 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 3. RESULTS AND DISCUSSION Collection of Entomopathogenic fungi of Sugarcane shoot and stem borer Sugarcane shoot and stem borer were collected from six sugarcane growing areas in Suphan Buri Province. Of the six areas, three areas were in Ban Nong Bua, Chorakhe Sam Phan Subdistrict, U Thong District where is a sugarcane plantation had been changed into rice fields for the first year and the other three areas were in Ban Nong Bo, Nong Bo Subdistrict, Song Phi Nong District where is a sugarcane planting that rotates sugarcane and rice according to the price of each year's production. All of the six sugarcane growing areas were infested with three species of sugarcane shoot and stem borer larva, namely, C. tumidicostalis (Hampson), C. sacchariphagus (Bojer) and S. inferens (Walker). Three cadaver larva specimens of C. tumidicostalis were found in sugarcane stalk. The first specimen cadaver larva of C. tumidicostalis had yellowish synnemata which come out of the space from the head and annal regions and through the respiratory tract of the larva. The synnemata produced white conidia at the tips (Figure 1). The second specimen cadaver larva of C. tumidicostalis has produced basal part white synnemata arising from all over body of the larva and produced grayish conidia at tips synnemata (Figure 2). On the other hand, the third specimen cadaver larva of C. tumidicostalis produced white mycelia which covered the larva and mass green conidia powdery after adding moisture (Figure 3). Figure 1 Characterization cadaver and synnemata of the first specimen. Figure 2 Characterization cadaver and synnemata of the second specimen. Figure 3 Characterization cadaver and conidia of the third specimen.
66 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Isolation and Identification of entomopathogenic fungi After the conidia and fruiting bodies of each fungus were cultured on a PDA medium and the fungi were purified, it was found that there were 3 distinct morphological characteristics. The first specimen cadaver larva of C. tumidicostalis had grown white colonies with a flat powdery consistency, extended radial growth, violet color, and changed to pink. However, when you flip over to look at the bottom of the petri dish, its appearance was gray and white. Although, the color of the culture medium did not change (Figure 4a-b). Subsequently, the conidia and conidiophore were put under a microscope and their appearance was observed. The conidiophore is colorless, well developed and not branched with the distal part producing 2 to many phialides. The phialide is bulging at the base and gradually narrows towards the tip to a long beak. The conidium joins in a chain from the ends of the phialide. Conidia are spherical, oval-like, and single-celled (Figure 5a-b). The first specimen was characterized as Paecilomyces lilacinus (Thom) Samson. Most recently, P. lilacinus was transferred to a new genus in Ophiocordycipitaceae as Purpureocillium lilacinum. The generic name refers to the purple conidia produced by the fungus [12]. A previous study observed a white edge of the mycelia at the beginning of the experiment but gradually turns into a round and bulging purple colonies. However, the conidia were also observed to oval to spindle and single celled as observed in this study [21]. It has code PSS. Figure 4. Colony characterization of Purpureocillium lilacinum. (a) upper, (b) reverse Figure 5. Morphological characteristion of Purpureocillium lilacinum. (a) conidium and phialid, (b) conidia. Bars are 20 micrometers The second specimen cadaver larva of C. tumidicostalis had fluffy white filament but when flipped over to look at the bottom of the petri dish it showed characteristics of orange germs that cause the color of the culture medium to turn orange (Figure 6a-b). Conidia are microconidia with one or two cells, round to oval or curved. Conidia form phialides that grow from the sides of the fiber or branching from conidiophores, short lines. The mature macroconidia are 3-5 septa, mostly curved at the tip, slightly elongated, straight, and varying in size. Macroconidia are made up of phialides that grow laterally from fibers (Figure 7a-b). Previously, it was observed that Fusarium spp. displayed two morphotypes with slightly different features showing branched and single long micro-phialides in morphotype 1 and 2, respectively. In addition, the septa were between 3- 5 as observed in this study in the case of morphotype 1,
67 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE however, for morphotype 2, the septa were between 3-7 [6]. In another study, the microconidia of Fusarium sp. shows similarity to the ones observed in this experiment The second specimen was identified as Fusarium sp. [10]. It has code FSS. Figure 6. Colony characteristion of Fusarium sp. (a) upper, (b) reverse Figure 7. Morphological characterization of Fusarium sp. (a) conidium and phialid, (b) conidia. Bars are 20 micrometers The third specimen cadaver larva of C. tumidicostalis had cottony and powdery consistency, radial and plane growth, the color varies between olive green, yellow and present a change of color from opaque yellow to light brown. When the petri dish is flipped, the bottom displays yellow color, however, the color of the culture medium did not change (Figure 8a-b). Conidia are cylindrical, single cells, often connected in long chains and present septate hyphae. The branched conidiophores with cylindrical phialides become thin toward the tip. (Figure 9a-b) This specimen was characterized as Metarhizium anisopliae. It has code MSS. The results resemble the results obtained in a previous study that showed compact green clusters of conidiophores characterizing Metarhizium spp. [2], [3]. Figure 8. Colony characteristion of Metarhizium anisopliae. (a) upper, (b) reverse
68 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Figure 9. Morphological characteristion of Metarhizium anisopliae. (a) conidium and phialid, (b) conidia. Bars are 20 micrometers Form the literature that we have perused, this fungal infection of C. tumidicostalis has not been reported in Thailand. This study is therefore the first report on the pathogenesis of entomopathogenic fungi of C. tumidicostalis larva, which will be used as a database for further researchers. Pathogenicity of entomopathogenic fungi against Chilo tumidicostalis larva in laboratory The drops of the conidia suspension of Fusarium sp., P. lilacinum and M. anisopliae was 5 μl at the concentration of 1x108 conidia/ml were made on the dorsal of the prothorax of C. tumidicostalis larva. The set-up had 10 larvae per treatment, 4 repetitions, with fresh cane cut into pieces as food which was changed every 2 days. It was incubated at 25±2°C for 10 days. After 10 days, it was found that the three isolates of entomopathogenic fungi were able to infect C. tumidicostalis after 2 days of inoculation. All three isolated entomopathogenic fungi, Fusarium sp. FSS, P. lilacinum PSS, and M. anisopliae MSS, showed pathogenicity against C. tumidicostalis larvae. The mortality rates and infestation times varied among the fungal isolates. M. anisopliae MSS exhibited the highest mortality rate, with an average of 87.50%±5.00% larval mortality. Fusarium sp. FSS and P. lilacinum PSS showed lower mortality rates, with 50.00%±8.16% and 40.00%±14.14% larval mortality, respectively. In terms of infestation time, M. anisopliae MSS had the fastest infestation, with an LT50 value of 1.003 days. Fusarium sp. FSS and P. lilacinum PSS had longer infestation times, with LT50 values of 7.77 days and 13.06 days, respectively. (Table 2). Table 2. Mortality of fourth instar larvae of Chilo tumidicostalis 10 days after treatment with different concentration and their median lethal time (LT50) Characteristics of the C. tumidicostalis larva that were infected with each species of fungi that cause disease. It was found that after placing the larva of C. tumidicostalis that were dead due entomopathogenic fungi, and incubated in the humidity box, they displayed the following characteristics: the Fusarium sp. showed that the mycelia covered larva after 7 days and produced synnemata after 14 days (Figure 10). Fusarium sp. been found to behave in this manner culturally in previous studies. They eventually produce synnemata which is one of its distinctive features of identification [5], [6]. Furthermore, they have been reported to cause favorable mortality against insect pests and have displayed features of agricultural pest control that are environmentally friendly. In addition, they have shown abundant sporulation which is a potential feature in commercial utilization as a biocontrol agent [18], [19]. When larva that was infected by P. lilacinum was placed in humidified boxes, the C. tumidicostalis larva began to dry and
69 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE mycelium formed after 3 days. However, after 6 days, purple-gray conidia were produced covering the larva (Figure 11). These symptoms have also been observed when this entomopathogenic fungi was being studied for the control of apple rust mite [4], [17]. On the other hand, the C. tumidicostalis larva that was infected with M. anisopliae began to produce white mycelium after 3 days of humidification and after 5 days the fungi began to form green conidia throughout the larva (Figure 12). Earlier studies on the morphology of M. anisopliae have described the development of white mycelia at the beginning and then changes into green after a few days which is similar to what was observed in this case ([7], [11], [16], [22]. Figure 10. Characteristion of C. tumidicostalis larva after inoculation with each entomopathogenic fungi 4. CONCLUSIONS The study identified three entomopathogenic fungi, Fusarium sp. FSS, P. lilacinum PSS, and M. anisopliae MSS, isolated from C. tumidicostalis larvae, a species of SSSBs. These fungi exhibited pathogenicity against the larvae, causing mortality. Among the isolates, M. anisopliae MSS showed the highest mortality rate and fastest infestation time, indicating its potential as a biological agent for the control of SSSBs. Further research and development are needed to optimize the use of these entomopathogenic fungi in integrated pest management strategies for sustainable sugarcane production. 5. ACKNOWLEDGEMENT Thanks to the National Biological Control Research Center (NBCRC), Central Regional Center, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom for supporting the entomopathogenic fungi of insects. and generously provided a place to conduct the experiment and completed this study. 6. REFERENCES [1] Abbott, W.S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267. [2] Bich, G. A., Castrillo, M. L., Kramer, F. L., Villalba, L. L., & Zapat, P. D. (2021). Morphological and Molecular Identification of Entomopathogenic Fungi from Agricultural and Forestry Crops. Floresta e Ambiente, 28(2), 1–11. https://doi.org/10.1590/2179-8087-FLORAM-2018-0086 [3] Chen, W. H., Han, Y. F., Liang, J. D., & Liang, Z. Q. (2019). Morphological and phylogenetic characterization of novel Metarhizium species in Guizhou, China. Phytotaxa, 419(2), 189–196. https://doi.org/10.11646/ phytotaxa.419.2.5 [4] Demirci, F. and E. Denizhan. 2010. Paecilomyces lilacinus, a potential biocontrol agent on apple rust mite Aculus schlechtendali and interactions with some fungicides in vitro. Phytoparasitica 38(2), 125–132. [5] Dongzhen, F., L. Xilin, C. Xiaorong, Y. Wenwu, H. Yunlu, C. Yi, C. Jia, L. Zhimin, G. Litao, W. Tuhong, J. Xu and G. Chunsheng. (2020). Fusarium Species and Fusarium oxysporum Species Complex Genotypes Associated with Yam Wilt in South-Central China. Frontiers in Microbiology 11, 1964. [6] Hafizi, R., Salleh, B., & Latiffah, Z. (2013). Morphological and molecular characterization of Fusarium. solani and F. oxysporum associated with crown disease of oil palm. Brazilian Journal of Microbiology, 44(3), 959. https://doi.org/10.1590/S1517-83822013000300047 [7] Hernández-Rosas, F., Andrés García-Pacheco, L., Figueroa-Rodríguez, K. A., Figueroa-Sandoval, B., Salinas Ruiz, J., Sangerman-Jarquín, D. M., & Liliana Díaz-Sánchez, E. (2016). Analysis of research on Metarhizium anisopliae in the last 40 years. Revista Mexicana de Ciencias Agrícolas Special Publication Number, 22. www. scopus.com [8] Humber, R. A. (2005). Entomopathogenic fungal identification, key to major genera. www.ars.usda.gov/ SP2UserFiles/Place/19070510/APSwkshoprev.pdf (October 21, 2015). [9] Kokkan, P., Siri, N., and Pitaksa N. (2008). The Effect of Stem Borers Infestations on Yield and Quality of 8 Sugarcane Varieties. khon kaen agriculture journal. 36 (Supplement Issue ): 165-171.
70 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE [10] Lacey, L. 1997. Manual of Techniques in Insect Pathology. Academic Press. 409 pp. [11] Liu, H., & Bauer, L. S. (2006). Susceptibility of Agrilus planipennis (Coleoptera: Buprestidae) to Beauveria bassiana and Metarhizium anisopliae. In J. Econ. Entomol (Vol. 99, Issue 4). http://jee.oxfordjournals.org/ [12] Luangsa-ard, J.J., Tasanathai, K., Mongkolsamrit, S. and Hywel-Jones, N. (2012) Atlas of Invertebrate pathogenic fungi of Thailand Volume 4. National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 102 pp. [13] Maneerat, T. and Suasa-ard, W. (2015). Population Trends of Sugarcane Moth Borers and Their Larval Parasitoid, Cotesia flavipes Cameron (Hymenoptera: Braconidae) in Growing Sugarcane Plantations. Kasetsart Journal (Natural Science) 49 : 403 – 412. [14] Mekapogu, A.R. (2021). Finney’s probit analysis spreadsheet calculator (Version 2021). https://probitanalysis. wordpress.com/ [15] Quinlan, R.J. (1988). Use of fungi to control insects in glasshouses. Pp. 19-36. In: M.N. Burge, (ed). Fungi in Biological Control Systems. Manchester University Press Manchester, UK. [16] Ramanujam, B., Poornesha, B., & Yatish, K. R. (2016). Screening of Beauveria bassiana and Metarhizium anisopliae Isolates against Sesamia inferens (Walker) . Indian Journal of Entomology, 78(4), 388. https://doi. org/10.5958/0974-8172.2016.00102.4 [17] Rumbos, C. I., A. Mendoza and S. Kiewnick. (2007). Effect of Paecilomyces lilacinus strain 251 on the survival and virulence of entomopathogenic nematodes under laboratory conditions. Nematologia Mediterranea 35(2): 103-107. [18] Santos, A. C. da S., A. G. Diniz, P. V. Tiago and N. T. de. Oliveira. (2020). Entomopathogenic Fusarium species: a review of their potential for the biological control of insects, implications and prospects. Fungal Biology Reviews 34(1), 41–57. [19] Sharma, L. and G. Marques. (2018). Fusarium, an Entomopathogen—A Myth or Reality?. Pathogens 7(4): 93. 1-15. [20] Suasa-ard, W. (2010). Natural Enemies of Important Insect Pests of Field Crops and Utilization as Biological Control Agents in Thailand, in Proceedings of International Seminar on Enhancement of Functional Biodiversity Relevant to Sustainable Food Production in ASPAC: In association with MARCO, November 9-11, 2010, Tsukuba, Japan. 1-15. [21] Sun, T., Wu, J., & Ali, S. (2021). Morphological and molecular identification of four Purpureocillium isolates and evaluating their efficacy against the sweet potato whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae). Egyptian Journal of Biological Pest Control, 31(1), 1–9. https://doi.org/10.1186/ S41938-021-00372-Y/FIGURES/7 [22] Zoberi, M. H. (1995). Metarhizium anisopliae, a Fungal Pathogen of Reticulitermes flavipes (Isoptera: Rhinotermitidae). Mycologia, 2(2), 49–50. https://doi.org/10.1080/002755 14.1942.12020904
71 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Pruchya Ekkathin1 *, Charanya Pinsupha2 , Yurawan Anantamanee3 , Phakasinee Klaimala⁴, Prachathipat Pongpinyo⁴ ¹Weed Research Group Research and Development Office of Plant Protection ²Renewable Energy Agronomy Research Institute ³Pest Management Group Research and Development Office of Plant Protection ⁴Agricultural Pesticide Research Group Research and Development Division of Agricultural Factors Correspondence to: Weed Research Group Research and Development Office of Plant Protection, Department of Agriculture, 50 Phaholyothin Road, Ladyao, Chatuchak, Bangkok 10900. E-mail [email protected] ABSTRACT: Study on efficacy of herbicides in sugarcane for alternative herbicides and safety crop production system the experiment were conduce in the glasshouse at weed research group department of agriculture. By testing herbicides spraying before weeds germinate (pre-emergence herbicides), such as amicarbazone, atrazine, diclozulam, diuron, fumiozaxin, hexazinone, imazapic, indaziflam,pendimethalin, s-metolachlor, sulfentarzone, metribuzin pendimethalin+imazapic, pendimethalin+amicarbazone, diuron+s-metolachlor, indaziflam ,metribuzin, atrazine, diuron, hexazinone+diuron herbicides spraying after weeds germinate (post-emergence herbicides) such as ametryn, diuron, bromacil, hexazinone, sulfentrazone, halosulfuron+ametryn,topamezone+diuron,triclopyr, glufosinate, 2,4-D/picolam+fluazifop, fluazifop-P-butyl+2,4-D, fluazifop-P-butyl+flumioxazin, glufosinate, diquat. Data were collected on the toxicity of herbicides on sugarcane, weed control efficacy and soil samples were collected for herbicide residue analysis at 2 months after herbicide application. The experimental results showed that pre-emergent herbicide that is no toxic to sugarcane and was effective in controlling weeds and found no residues in the soil, namely hexazinone+diuron at the rate of 2062.5 g(ai)/hectare. As for post emergence namely halosulfuron+ametron, at the rate of 56.25+2,500 g(ai)/hectare. These herbicides can control narrow-leaved weeds such as Digitaria ciliaris, Brachiaria reptans and Echinochloa colona, and broadleaved weeds such as Euphorbia heterophylla, Camonea umbellata and Trinathema portulacastrum. Therefore, herbicides hexazinone+diuron at the rate of 2062.5 g(ai)/hectare and halosulfuron+ametron at the rate of 56.25+2,500 g(ai)/ hectare to expand the experimental results in the field of sugarcane conditions. Keyword: herbicide efficiency, sugarcane toxicity, alternative herbicides and safety P-001 Study on Efficacy of Herbicides in Sugarcane for alternative herbicides and safety crop production system
72 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Jariya Roddee1 , Jureeemat Wangkeeree2 and Yupa Hanboonsong3 1 School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, Nakhon Ratchasima, Thailand ²Department of Agricultural Technology, Faculty of Science and Technology, Thammasat University Rangsit Centre, Khlong Nueng, Klong Luang, Pathum Thani, Thailand ³Department of Entomology and Plant Pathology, Faculty of Agriculture, Khon Kaen University, Nai Muang, Muang, Khon Kaen, Thailand *Correspondence to: School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, Nakhon Ratchasima, Thailand. E-mail [email protected] ABSTRACT: Chemical control is the most used method for managing Matssumuratettix hiroglyphicus, the vector of the phloem-limited phytoplasma with sugarcane white leaf disease. The objectives of this study were to determine the influence of soil-drench applications of neonicotinoids (imidacloprid) on the probing behavior of M. hiroglyphicus on sugarcane, using the electrical penetration graph (EPG) technique for monitoring the feeding behavior of M. hiroglyphicus after application of imidacloprid on sugarcane 1, 7, 14, 35, and 60 days, compared with the control (without the chemical). The drenched applications of imidacloprid on sugarcane disrupt the M. hiroglyphicus probing. The mean duration of the probing waveform was reduced by a significant difference (P< 0.05) at 7, 14, 35, and 60 days. Moreover, the total duration of waveforms C and D was reduced significantly with the control group. EPG variables related to phloem sap ingestion, with a significant reduction (70%) in the duration of this activity compared to untreated plants. This study demonstrates soil-applied imidacloprid's interference on the M. hiroglyphicus feeding behavior and mortality on sugarcane. Therefore, the imidacloprid influenced the feeding behavior and mortality of M. hiroglyphicus leafhoppers vector, of sugarcane white leaf disease. These findings recommend that the residual effect of drench application of imidacloprid longer than 35 days is a useful strategy for M. hiroglyphicus management. Keywords: electropenetrography (EPG), feeding behavior, imidacloprid, phloem sap ingestion, mortality P-002 Evaluating the effect of imidacloprid on the feeding behavior of leafhopper Matssumuratettix hiroglyphicus (Hemiptera: Cicadellidae) vector of sugarcane white leaf disease
73 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION Leafhopper Matssumuratettix hiroglyphicus (Hemiptera: Cicadellidae) vector of sugarcane white leaf (SCWL) disease on sugarcane. The SCWL phytoplasma is an obligated parasite of plant and insect cells (Nakashima et al., 1993; Wongkaew et al., 1997) and cannot be cultured (Hanboonsong et al., 2002, 2006). The phytoplasma transmits in a persistent propagative manner; therefore, the microorganism reproduces in the insect, and infected vectors remain able to transmit the pathogen during their entire life (Hogenhout et al., 2008; Roddee et al., 2019). The leafhopper M. hiroglyphicus has transmission from generation to next generation by eggs (transovarial transmission) (Hanboosong et al., 2002) and they also were phytoplasma is widely distributed in the insect’s body (Roddee et al., 2019). The transmission type is called persistent propagative or circulative propagative including acquisition access period (AAP), Incubation period (LP) or latent period, and inoculation access period (IAP). The feeding duration necessary to acquire a sufficient titer of phytoplasma is the AAP. The time that elapses from initial acquisition to the ability to transmit phytoplasma is known as the LP and the minimum time required for an infective vector from the latent period until inoculation to the host plant is called the inoculation access period (Weintraub and Beanland, 2006). Current control methods for vector-borne diseases rely mainly on insecticides in the propagation field. One of the world’s best-selling insecticides is imidacloprid, 1-[(6-Chloro-3-pyridinyl) methyl]-N-nitro-2-imidazolidinimine which belongs to the chemical group of neonicotinoid insecticides. Neonicotinoids have selective toxicity for insects and act by binding to the nicotinic acetylcholine (ACh) receptors in the receiving nerve cells of the central nervous system (Jeschke, and Nauen 2008). This insecticide has systemic activity (Barry et al. 2014), and it is readily taken up by plant roots and acropetally translocated via the xylem vascular system. Imidacloprid was followed in 1999 by thiamethoxam (Maienfisch et al. 2001) and clothianidin, a metabolite of thiamethoxam (Meredith et al. 2002). An innovative technology known as “electrical penetration graph (EPG)” (McLean and Kinsey 1964, Backus and Bennett 2009) is a very useful tool to assess stylet probing behavior of hemipterans and other piercing–sucking insects, (Tjallingii et al. 2010; Butler et al. 2012; Jacobson and Kennedy 2013, 2014; Civolani et al. 2014; Pearson et al. 2014). Electropenetrography (EPGs) has been used as an effective tool to study the feeding behaviors of sucking insects. (McLean and Kinsey (1964) first developed an alternating current (AC)-based EPG technique to monitor the style penetration behavior of an aphid. A direct current (DC)- Tjallingii later developed based EPG technique, Tjallingii (1978) which provided more detailed waveforms than the AC-EPG. EPGs has been successfully used to assess the effects of insecticides on the feeding behavior of piercing–sucking insect pests, including the rice leafhopper [Nilaparvata lugens (Stål)], [Sogatella furcifera (Horvath)], [Laodelphax striatellus (Fallen)] aphids [Aphis fabae Scopoli], [Aphis gossypii (Glove)] (Harrewijn and Kayser, 1997) and glassy wing sharpshooter [Homalodisca coagulate (Say)] (Bextine et al., 2004), psyllids, whiteflies, aphids, and thrips (Jiang et al. 2000; Butler et al. 2012; Jacobson and Kennedy 2013, 2014; Serikawa et al. 2013; Civolani et al. 2014). The major objective of this study was to use EPG technology to assess the effects of imidacloprid on the feeding behavior of the leafhopper M. hiroglyphicus on sugarcane. 2. MATERIALS AND METHODS 2.1 Insect colonies Insect colonies of leafhopper M. hiroglyphicus used in the study were collected in 2021 from an experimental sugarcane at Nakorn Ratchasima and were reared continuously on sugarcane seedlings in the laboratory without exposure to any insecticides. Sugarcane plants were grown at 28 ˚C ± 1˚C and a photoperiod of 12:12 h (L:D). And the insects were maintained in the laboratory at 28 ˚C ± 1˚C and 12:12 h (L:D). 2.2 Plants Material Plants used in the experiment were treated with soil applications of imidacloprid at a rate of 0.61 g of product per 1 L. of water. The plants were sprayed with insecticide until runoff. The treated plants were left to dry for 1 h and then kept in a greenhouse for 24h before the mortality and EPG recordings were checked. Untreated control plants were also included in the trial to serve as controls. 2.3 The Electropenetrophapy for feeding behavior of M. hiroglyphicus The leafhopper adult EPG recordings, the leafhopper on sugarcane leave of insecticide-treated and untreated plants were recorded in a Faraday cage using a Giga-8 DC–EPG with a 109 input resistance (Wageningen Agricultural University, Wageningen, The Netherlands). One sugarcane seedling in the tillering stage was transplanted in each soil pot. EPG recordings were made 1, 7, 14, 35, and 60 days after treatment. Before recording, leafhoppers were starved for 0.5 h. The total number of waveform or probing events and time duration spent in intercellular stylet penetration or pathway phase (waveform A); salivation into phloem sieve elements (waveform C); and phloem sap ingestion (waveform D) were recorded. EPG was recorded for 3 hours by Stylet+ software and using the MS Excel workbook
74 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE for automatic parameter calculation of EPG data and Probe 3.0 Windows software developed by the Department of Entomology, Wageningen, The Netherlands. Data from 1–20 adult leafhoppers were recorded for each treatment. The duration and number of probes for each EPG waveform represent the mean value of the sum of durations (waveform duration per insect, WDI; Backus et al. 2009) and number of occurred times of all occurrences (number of waveform events per insect, NWEI) of the waveform within the observation time. 2.4 Data analysis Statistical analysis EPG data were statistically analyzed with SAS (1990). Proc GLM procedures were used for variance analyses. Percentage of each wave-Data were log- and square root-transformed before analysis of variance (ANOVA) to reduce variability and improve homogeneity. 3. RESULTS AND DISCUSSION The EPG waveforms of the leafhopper showed characteristics and were classified into several distinctive waveforms based on the patterns observed by Roddee et al. (2017; 2021). These waveforms are Np for nonpenetration, A, and B for penetration initiation and pathway phase, C for salivation and stylet movement, D for intracellular activity in the phloem tissue, phloem sap ingestion, and stylets in the phloem tissue (Figure 1). Several studies have demonstrated that EPG technology can successfully be used to assess the effects of insecticides on the feeding behavior of piercing–sucking insect pests and disease transmission, including psyllids (Butler et al. 2012, Serikawa et al. 2013 ), whiteflies (Jiang et al. 2000, Civolani et al. 2014), aphids (Jacobson and Kennedy 2014), and thrips (Jacobson and Kennedy 2013). Insecticides that produce rapid feeding cessation of insect vectors by affecting muscle contraction and inducing partial paralysis can also reduce the transmission of diseases by reducing pest movement and spread of these vectors within and between fields, reducing vector fecundity and fertility, and preventing colonization of pest populations. In the current study, EPG was used to examine the feeding behavior of the leafhopper M. hiroglyphicus when feeding on sugarcane plants treated with imidacloprid insecticides. During the 3 hours recording period, imidacloprid significantly (P< 0.05) altered the feeding behavior of leafhoppers compared with untreated control plants. The average number of probe events during stylet on plant tissue significantly differed on 60 days imidacloprid-treated plants compared to 1-day imidacloprid-treated and untreated plants (Figure 2). The probing number of the leafhopper M. hiroglyphicus on the control imidacloprid-treated plants and 1-day imidacloprid-treated plants frequently more significantly (P< 0.05) than 7, 14, 35, and 60 days imidacloprid-treated plants. Similarly, the average time during the probe significantly differed (P< 0.05) on 7, 14, 35, and 60 days of imidacloprid-treated plants compared with the untreated control (Figure 3). Figure 1. Characteristic EPG waveforms recorded from M. hiroglyphicus: Np, non-probe/non-penetration, A, and B; pathway phase, C; salivation, D; phloem sap ingestion.
75 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Waveforms C and D, the leafhopper M. hiroglyphicus on the control imidacloprid-treated plants and 1-day imidacloprid-treated plants fed significantly (P< 0.05) higher than 7, 14, 35, and 60 days imidacloprid-treated plants (Figure 2). Similarly, the average time during waveform C significantly differed on 14, 35, and 60 days of imidacloprid-treated plants compared with the untreated control (Figure 3). The average time during waveform D significantly differed (P< 0.05) on 7, 14, 35, and 60 days of imidacloprid-treated plants compared with the untreated control (Figure 3). Moreover, the number of probes on waveform C and D were reduced from 7 days after imidacloprid-treated to 14 after days imidacloprid-treated and increased from 35 days after imidacloprid-treated to 60 after days imidacloprid-treated. From these, the imidacloprid was more effective on the leafhopper M. hiroglyphicus 14 days after imidacloprid-treated. Insecticides can reduce plant pathogen transmission by direct insect vector mortality and reduction in feeding. Most insecticides do not hamper the feeding of insect vectors before death, allowing pathogen transmission with related salivation and ingestion waveforms (Jiang et al., 2000). The reduction in efficiency of pathogen transmission by insect vectors depends on how actively insecticides prevent insects from accessing the phloem or xylem tissue (Jiang et al., 2000). Figure 2. Average number of waveform event per insect for stylets probing behavior in 3 hours by the leafhopper M. hiroglyphicus on the sugarcane plants treated with imidacloprid and untreated control plants. Figure 3. Mean waveform duration per insect for stylets probing behavior in 3 hours by the leafhopper M. hiroglyphicus on the sugarcane plants treated with imidacloprid and untreated control plants. The imidacloprid-treated plants were affected by the duration of probing behavior, after imidacloprid-treated on sugarcane the probing was reduced from 1-day imidacloprid-treated to 60 days imidacloprid-treated (Figure 4). Using EPG, several studies have also shown that cyantraniliprole and imidacloprid insecticides significantly reduced the total amount of time spent probing as well as the number of phloem-feeding and intracellular and intercellular stylet
76 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE penetration events by potato psyllids (Butler et al. 2012), aphids (Jacobson and Kennedy 2014), thrips (Jacobson and Kennedy 2013), and whiteflies (Civolani et al. 2014). These studies concluded that these classes of insecticides could potentially play an important role in preventing the transmission of plant pathogens associated with these insect pests. Figure 4. Percentage of individuals having a certain EPG waveform in all tested leafhopper M. hiroglyphicus samples. 4. CONCLUSION In conclusion, the results of the present EPG study clearly indicated that imidacloprid significantly deterred the probing behavior of the M. hiroglyphicus leafhoppers vector, of sugarcane white leaf disease. The imidacloprid influenced the feeding behavior and mortality of the M. hiroglyphicus leafhoppers. These findings recommend that the residual effect of drench application of imidacloprid longer than 35 days is a useful strategy for M. hiroglyphicus management. Thus, similar to imidacloprid could become an important tool for sugarcane farmers to effectively managing the leafhoppers vector and potentially reduce the transmission and spread of sugarcane white leaf disease. 5. ACKNOWLEDGMENT The authors would like to express appreciation for the support of the sponsors SUT Research and Development Fund [Project Number = SUT3-302-62-12-27]. 6. REFERENCES Backus, E. A. and Bennett, W. H. (2009). The AC-DC correlation monitor: new EPG design with flexible input resistors to detect both R and emf components for any piercing sucking hemipteran. Journal of insect physiology, 55: 869– 884. Barry, J. D., Portillo, H. E., AnnanI, B., Cameron, R. A., Clagg, D. G., Dietrich, R. F., Watson, L., J., Leighty, R. M., Ryan, D. L. and McMillan, J. A. (2014). Movement of cyantraniliprole in plants after foliar applications and its impact on the control of sucking and chewing insects. Pest Management Science, 71: 395– 403. Bextine, B., Harshman, D. Johnson, M., and Miller. T. (2004). Impact of pymetrozine on glassy-winged sharpshooter feeding behavior and rate of Xylella fastidiosa transmission. Journal of Insect Science, 4: 1-6. Butler, C. D. Walker, G. P. Trumble, J. T. (2012). Feeding disruption of potato psyllid, Bactericera cockerelli, by imidacloprid as measured by electrical penetration graphs. Entomologia Experimentalis et Applicata. 142: 247 – 257. Civolani, S., Cassanelli, S., Chicca, M., Rison, J. L., Bassi, A., Alvarez, J. M., Annan, I. B., Parrella, G., Giorgini, M. and Fano, E. A. (2014). An EPG study of the probing behavior of adult Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae) following exposure to cyantraniliprole Journal of Economic Entomology. 107: 910 – 919.
77 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Hanboonsong, Y., Choosai, C., Panyim, S., and Damak, S. (2002). Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura). Insect Moleculae Biology. 11: 97–103. Hanboonsong, Y., Ritthison, W., Choosai, C., and Sirithorn, P.(2006). Transmission of sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector. Journal of Economic Entomology. 99: 1531–1537. Hogenhout, S. A., Oshima, K., Ammar, E. D., Kakizawa, S., Kingdom, H. N., and Namba, S. (2008). Phytoplasmas: bacteria that manipulate plants and insects. Molecular Plant Pathology 9: 403–423. Harrewijn, P., and Kayser, H. (1997). Pymetrozine, a fast-acting and selective inhibitor of aphid feeding. In-situstudies with electronic monitoring of feeding behaviour. Pesticide science49: 130-140. Jacobson, A. L. and Kennedy, G. G. (2014). Electrical penetration graph studies to investigate the effects of cyantraniliprole on feeding behavior of Myzus persicae (Hemiptera: Aphididae) on Capsicum annuum. Pest Management Science. 70: 836 – 840. Jacobson, A. L. and Kennedy, G. G. (2013). Effect of cyantraniliprole on feeding behavior and virus transmission of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) on Capsicum annuum. Crop Prot. 54: 251– 258. Jeschke, P. and Nauen, R. (2008). Neonicotinoids—from zero to hero in insecticide chemistry. Pest management science. 64: 1084–1098. Jiang, Y. X., De Blas, C., Barrios, L. and Fereres, A. (2000). Correlation between whitefly (Homoptera: Aleyrodidae) feeding behavior and transmission of tomato yellow leaf curl virus. Annals of the Entomological Society of America. 9: 573 – 579. Maienfisch, P., Huerlimann, H., Rindlisbacher, A., Gsell, L., Dettwiler, H., Haettenschwiler, J., Sieger, E. and Walti, M. (2001). The discovery of thiamethoxam: a second-generation neonicotinoid. Pest management science. 57:165–176. McLean, D.L. and Kinsey, M.G. (1964). A technique for electronically recording aphid feeding and salivation. Nature 202: 1358– 1359 Meredith, R.H., Heatherington, P.J. and Morris, D.B. (2002). Clothianidin—a new chloronicotinyl seed treatment for use on sugar beet and cereals: field trial experiences from Northern Europe. The BCPC Conference: Pests and diseases, Volumes 1 and 2. Proceedings of an international conference held at the Brighton Hilton Metropole Hotel, Brighton, UK, 18–21 November 2002. British Crop ProtectionCrop Protection Council. pp. 691–696. Nakashima, K., Kato, S., Iwanami, S., and Murata, N.(1993). DNA probes reveal relatedness of rice yellow dwarf mycoplasmalike organisms (MLOs) and distinguish them from other MLOs. Applied and Environmental Microbiology. 59:1206–1212. Roddee J., Backus E. A., Wangkeeree, J., and Hanboonsong, Y. (2021). Alteration in the Stylet Probing Behavior and Host Preference of the Vector Matsumuratettix hiroglyphicus (Hemiptera: Cicadellidae) After Infection with Sugarcane White Leaf Phytoplasma. Journal of Economic Entomology.114 (3), 1081–1090. Roddee, J., Kobori, Y. and Hanboonsong, Y. (2019). Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). Entomologia Experimentalis et Applicata. 167: 108–117. Pearson, C. C., Backus, E. A., Shugart, H. J. and Munyaneza, J. E. (2014). Characterization and correlation of EPG waveforms of Bactericera cockerelli (Hemiptera: Triozidae): variability in waveform appearance in relation to applied signal. Annals of the Entomological Society of America.107: 650– 666. Serikawa, R. H., Backus, E. A. and Rogers, M. E. (2013). Probing behavior of adult Asian citrus psyllid (Hemiptera; Liviidae) are not appreciably affected by soil application of field-rate aldicarb to citrus. Florida Entomologist. 96; 1334 – 1342. Tjallingii, W.F. (1978). Electronic recording of penetration behavior by aphids. Entomol Exp Appl 24: 521– 530. Tjallingii, W. F., Garzo, E. and Fereres, A. (2010). New structure in cell puncture activities by aphid stylets: A dual-mode EPG study. Entomologia Experimentalis et Applicata. 135: 193– 207 Weintraub, P. G. and Beanland, L. (2006). Insect vectors of phytoplasmas. Annual review of entomology. 51: 91–111. Wongkaew, P., Hanboonsong, Y., Sirithorn, P., Choosai, C., Boonkrong, S., Tinnangwattana, T., Kitchareonpanya, R. and Damak, S. (1997). Differentiation of phytoplasmas associated with sugarcane and gramineous weed white leaf disease and sugarcane grassy shoot disease by RFLP and sequencing. Theoretical and Applied Genetics. 95: 660–663.
78 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Weerakorn Saengsai1 *, Benjawan Rattawat¹ and Nattapat Khumla² ¹Khon Kaen Field Crops Research Center, Muang, Khon Kaen, 40000, Thailand. ²Nakhon Sawan Field Crops Research Center, Tak Fa, Nakhon Sawan, 60190, Thailand. *Correspondence to: [email protected] ABSTRACT: Water deficit is considered one of the most important limiting factors to crop productivity and growth rates. Plants can overcome this constraint by accumulating osmoprotectants to maintain cell water balance, which enables them to grow under water stress conditions. In this study, the expression of three genes, Proline Transporter (ProT), Trehalose phosphate synthase (TPS), and Sugar transporters (PST3), in sugarcane clones/varieties was investigated using qRT-PCR to better understand their drought tolerance mechanisms. The results revealed that all three genes were expressed in both stress and non-stress conditions. Moreover, the expression profiles of these genes varied across different sugarcane clones/ varieties, the NSUT13-154 and DOANakhon Sawan 1 demonstrated higher expression levels of these genes in dehydration compared to the control group. Physiological and biochemical changes related to oxidative stress, osmoprotectant substance accumulation, and cell destruction substances were also identified. Testing in a controlled environment, coupled with the detection of growth, physiological, and biochemical changes provided an effective approach for evaluating and selecting drought-tolerant sugarcane clones. The testing results showed that the tested sugarcane clones/varieties had low drought tolerance compared to Khon Kaen3. The DOA Nakhon Sawan 1 and NSUT13-154 potentially exhibited moderate water stress tolerance, while NSUT13-289 and LK92-11 were potentially water stress-sensitive. These findings have important implications for the development of new drought-tolerant sugarcane varieties. Keyword: Sugarcane, gene expression, drought, physiological and biochemical. P-003 Analysis of Gene Expression and Biochemical Changes under Controlled Conditions in Promising Sugarcane Clones Resistant to Drought
79 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION In 2015–2020, the average sugarcane production in Thailand was around 63 tons/hectare, with an average commercial cane sugar (CCS) of 12.41%. Currently, an estimated 330,000 sugarcane growers are actively cultivating sugarcane in Thailand [1].Environmental stresses pose significant limitations on plant growth and crop productivity. Sugarcane, a vital source of sugar and ethanol, is a crop with relatively high water requirements, making it highly susceptible to water scarcity [2]. Studies suggest that sugarcane typically produces between 8 and 12 tons of per megaliter (ML) of irrigation water, and water deficit can result in productivity losses of up to 60% [3]. Consequently, areas suitable for sugarcane production are often concentrated in regions with favorable rainfall patterns, while other regions require supplementary or full irrigation for successful crop cultivation [4]. Water availability in sugarcane plantations in Thailand is variable, and with less than 10% of the cultivated area being irrigated, is affected strongly by climate. Almost every year, sugarcane production in Thailand is affected by drought periods, causing an estimated annual average loss of 10 million tons of cane (about 10% of the national cane production) [1]. Drought frequently occurs in the sugarcane growing regions of Thailand, especially in the north-eastern and central regions. In 2019, the cumulative annual rainfall in Thailand was about 1,343 mm, a decrease of about 8% compared to 2018, and resulting in severe impact on the sugarcane production. Osmoprotectants are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume [5], and may play other roles regarding tolerance, as proteins stabilizing and antioxidant action [6]. They include sugars, mainly fructose and sucrose, sugar alcohols (like myo-inositol), complex sugars (like trehalose and fructans) and charged metabolites (like glycinebetaine, proline and ectoine) [5]. Gupta [7] using cDNA libraries associated to the qRT-PCR validation of drought related genes, revealed differences greater than 2-fold regarding the expression of given genes during dehydration stress. The most recent report was carried out by Iskandar [8] investigated whether the degree of expression of eight stress-related genes (P5CS, OAT, AS, PST5, TF1, LEA, POX and dehydrin) was correlated with the sucrose content in the sugarcane culm, and whether such genes were also responsive to water deficit stress. Almost all selected genes were up regulated, with exception of POX that was down regulated after water deficit stress. Also investigated the possible roles of proline and glycine betaine (GB) in mitigating the effect of chilling stress in the sprouting nodal buds of sugarcane [10]. To accomplish this evaluation, they performed a pre-treatment with proline bud chips and GB, obtaining a substantialreduction in the H2 O2 production and an increase in the synthesis efficiency of soluble sugars, protecting developing tissues from the effects of chilling stress. The objective of this study is to anlyze gene expression using qRT-PCR and employ statistical analysis as the primary tool for selecting reference genes. Furthermore, the study aims to compare the biochemical changes related to osmoprotectans under controlled conditions in promising sugarcane clones that exhibit resistance to drought [9]. 2. MATERIALS AND METHODS 2.1. Plant growth and water stress treatment Sugarcane plantlets of the cultivars KK3 (tolerant) and NSUT13-154, NSUT13-289, DOA Nakhon Sawan 1 and LK92-11 tested in located in Khon Kaen Field Crops Research Center, Khon Kaen province, Thailand, from October to December 2021, were cultivated under greenhouse conditions at 30 °C with 56% of relative humidity for 10 days in sterilized soil. The experiment was arranged in a completely randomized design, with three biological replicas for control and treated plants, which were sampled at three experimental time-points, totaling 25 plants for each cultivar. All plantlets were irrigated daily as described previously with 25% Hoagland solution. After 2 months of growth, the water stress was induced by irrigation suppression. Daily irrigation was maintained for control plants. The substrate water content was measured by gravimetric methods to observe the percentage of humidity in weight during the experimental treatment. Treated plants were submitted to increasing water deficiency and were collected Leaf water content was determined based on the method described by Silva [9]. Leaf disks from each plant were collected, immediately stored at -80 °C prior to use. 2.2. RNA extraction and cDNA synthesis Total RNA extraction was performed using the commercial GF-1 Reagent (Viviantis, Malaysia) following the manufacturer's instructions. For this, 30 mg of total RNA was used as a template to produce cDNA probes. Plant tissue (100 mg) was ground to a fine powder using a pre-chilled mortar and pestle in liquid nitrogen. Buffer TR or Buffer HR (400 µl) was added to the ground tissue and mixed thoroughly by vigorous vortexing. The sample was centrifuged at maximum speed for 3 minutes, and the lysate was transferred to a Homogenization column assembled in a collection tube. After centrifuging at maximum speed for 2 minutes, the flow-through was saved, and 350 µl of 80% ethanol was added to it. The sample (including any precipitate) was transferred to an RNA Binding Column assembled in a collection tube. After centrifugation at 10,000 x g for 1 minute, the flow-through was discarded. Wash Buffer (500 µl) was added and centrifuged at maximum speed for 1 minute. The flow-through was discarded,
80 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE and DNase I Digestion Mix (70 µl) was pipetted into the RNA Binding Column, followed by incubation at room temperature for 15 minutes. Inhibitor Removal Buffer (500 µl) was added and centrifuged at maximum speed for 1 minute, and the flow-through was discarded. The column was washed with 500 µl of Wash Buffer and centrifuged at 10,000 x g for 1 minute. This wash step was repeated, and the column was centrifuged at 10,000 x g for 1 minute to remove traces of buffer. The column was placed in a new microcentrifuge tube, and RNase-free Water (40-60 µl) was added directly onto the membrane and incubated for 1 minute. After centrifugation at 10,000 x g for 1 minute, the RNA was stored at -20°C or -80°C. To synthesize cDNA, the reagents were mixed well, and the tubes were briefly centrifuged. The RNA-primer mixture was prepared with 3 µl of total RNA, 1 µl of Oligo d(T)18, 1 µl of 10 mM dNTPs mix, and 5 µl of nuclease-free water. The mixture was incubated at 65°C for 5 minutes and chilled on ice for 2 minutes. After a brief spin-down, the cDNA Synthesis Mix was prepared with 2 µl of 10X Buffer M-MuLV, 100 units of M-MuLV Reverse Transcriptase, and nuclease-free water to make a total volume of 10 µl. Then, 10 µl of the cDNA synthesis Mix was added to each RNA-primer mixture, gently mixed, and briefly centrifuged. The mixture was incubated at 42°C for 60 minutes. The reaction was terminated by incubating the tubes at 85°C for 5 minutes. After chilling the tubes on ice and a brief centrifugation, the synthesized cDNA could be directly used for downstream applications or stored at -20°C. 2.3. Real time PCR The real time PCR reactions were performed with SYBR Green Master Mix (Roche Applied Science) in a LightCycler® 480 System thermocycler (Roche Applied Science). To normalize the relative expression of selected genes, proline transporter (ProT), trehalose phosphate synthase (TPS), and sugar transporters (PST3) were used as reference. Primers were designed in OligoAnalyzer 3.1 (http://eu.idtdna.com), randomly selected in different time points using Primer Express 3.0 software (Roche Applied Science). Primers sequences were searched against the KEGG pathway database using the Blast tool to verify the specificity of the sequences. The sequences and the expression data values observed for selected genes are detailed. For each selected gene three biological replicates were assayed independently. Each reaction was performed in duplicate using 2.0 ml of a 1:10 dilution of the sample cDNA, primersto a final concentration of 400 nM, 12.5 ml of SYBR Green Master Mix and PCR-grade water to a final volume of 20 ml. For negative controls, cDNA templates synthesized without Superscript II Reverse Transcriptase were used. PCR amplifications were carried out using 50 OC for 1 min, 94 OC for 5 min and 30 cycles consisting of 94 OC for 1 min and 72 OC for 1 min as run parameters. Analysis relative expression [11], Gene expression content was calculated as in the below equation. Ratio = 2-ΔΔ∆Ct ΔΔ∆Ct = (Ct Target – reference) sample - (Ct Target – reference) calibrator 2.4 Biochemistry test Determination of Biochemistry was made on the blade (leaf+1): the first completely expanded leaf from the apical "dewlap" or visible auricle [12].which is ideal for assessing biochemical compounds. To determine the levels of trehalose, 1 g of fresh leaf tissue was assessed by the enzymatic method described by Neves [13] and adapted by Queiroz and Santos [14]. The hydrolysis reaction was incubated in a water bath for 1 h at 60 ºC. Each mol of glucose represented 0.5 mol of trehalose. Fresh leaves (0.5 g) were ground with liquid nitrogen. Then, 10 ml of 3% sulphosalicylic acid was added and centrifuged at 1120× g for 20 min at 4 ºC, and 2 ml of ninhydrin acid and 2 ml of glacial acetic acid were added to the supernatant. The mixture was placed in a boiling water bath for 60 min, then removed and immediately cooled in ice bath for 5 min. Then, 4 ml of toluene was added, and the mixture was vortexed for 20 s. Absorbance was recorded at 520 nm. Proline concentration was determined using a proline calibration curve and expressed as µmol g−1 FW. The superoxide dismutase (SOD) activity was determined according to the method described by Beauchamp and Fridovich [15]. The extract was obtained from 300 mg of fresh leaf tissue, and 100 µl was taken from the vegetable extract and put into the reaction medium, and then the homogenate was centrifuged at 14,000 g for 15 min at 4 ºC. This medium was prepared with 50 mM potassium phosphate buffer (pH 7.8), 100 nM EDTA, 75 µM nitro blue tetrazolium (NBT), 13 mM methionine and 2 µM riboflavin, and final volume of 3 ml distilled water. Then, readings were taken by a spectrophotometer at 560 nm. Guaiacol peroxidase activity (GPX) was measured by reducing H2 O2 with oxidation of guaiacol following Chance and Maehly [16]. The reaction mixture was 1500 µl sodium buffer phosphate (100 mM) pH 7, 120 µl H2 O2 (15 mM), 480 µl guaiacol (20 mM), and 50 µl of enzyme extract. The absorbance raise was recorded at 470 nm. The GPX activity was defined as µM min−1 mg−1 FW or UA mg−1 FW.
81 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE The ascorbate peroxidase (APX) activity was determined according to the method described by Nakano and Asada [17] with some changes. The extract was obtained from 20 mg of fresh leaves, and 30 µl of the extract was taken and put into the reaction medium, and then the homogenate was centrifuged at 14,000 g for 15 min at 4 ºC. This medium was prepared with 50 mM potassium phosphate buffer (pH 7.5), 0.05 mM H2 O2 , and 0.5 mM sodium ascorbate, and the volume was completed with 3 mL of distilled water. Readings of absorbance were taken at 290 nm for 1 min, observing the decrease of ascorbate concentration. An ascorbate extinction coefficient of 2.8 mM-1 cm-1 at 290 nm was used. Soluble proteins were quantitatively determined following the method of Bradford [18]. The amount of malondialdehyde (MDA) was estimated following Heath and Packer [19]. A sample of 0.5 g of fresh leaves was extracted in 1.5 ml trichloroacetic acid 1% w/v (TCA) and centrifuged at 1120 × g for 10 min, and 1 ml thiobarbituric acid 0.1% w/v (TBA) was added to 0.5 ml of the supernatant. The mixture was boiled at 95 ºC for 30 min and cooled on ice for 15 min. Absorbance was recorded at 532 and 600 nm. MDA content was calculated as in the below equation. 2.5 Statistical analysis The t-test was used to estimate significant changes in the relative expression levels (p < 0.05). 3. RESULT AND DISCUSSION Osmotic stress is the prominent factor due to drought. Normally, plant cells have higher osmotic pressure than to soil, so it takes water and minerals from the soil but in case of drought stress the osmotic potential of soil increases by the high aggregation of water in the soil that makes plant unable to take water and essential minerals from the soil. This condition creates a condition of physiological drought in soil [20]. In present study, to determine whether the expression of ProT, TPS, and PST3 genes was responsive to abiotic stresses, the expression patterns of these genes in sugarcane were examined. Sugarcane 60 days showed the appearance of dwarf plants after 14 days of drought treatment. We measured the expression levels of sugarcane ProT, TPS, and PST3 genes after 14 days of drought stress; ProT genes had expression levels more than twice as high as before stress. TPS showed the greatest change in expression (Figure 1-3), The NSUT13-154 and DOA Nakhon Sawan 1 demonstrated higher expression levels of these genes in dehydration compared to the control group. We found that proline, trehalose, MDA and hydrogen peroxide accumulate more in tolerant verities rather than susceptible plant when expose to drought and its level further increases when we increase the EC of irrigation water respectively (Figure 5-7), control plant show less proline, trehalose, MDA and hydrogen peroxide in their cell that signifies that plant under stress condition amass more proline, trehalose, MDA and Hydrogen peroxide in the cell as compared to non-stressed plant. Morphologically, plants show various symptoms under drought conditions that truly prove the adverse effect of water on plant like plant growth reduction, decrease in length of internodes, cane girth and juice quality etc. [21]. Plants physiological and biochemical activity suffers due to disruption of anabolic and catabolic phenomenon [22]. Sugarcane plant has been categorized as moderately sensitive towards salt stress [23] and each plant or variety responds differently to drought stress due to their genotypic KK3 (tolerant) and NSUT13-154, NSUT13-289, DOA Nakhon Sawan 1 and LK92-11, that were evaluated for the effect of drought stress on various factors like proline, trehalose, MDA and hydrogen peroxide. The result revealed the increased accumulation of proline, trehalose, MDA and hydrogen peroxide activity can be used as biochemical markers for screening the efficient genotype of sugarcane for drought tolerance. Tolerant genotypes accumulate large amount of compatible solute that maintains the turgor pressure of cells. 5. CONCLUSIONS The study findings indicate that DOA Nakhon Sawan 1 and NSUT13-154 have the potential for moderate water stress tolerance in sugarcane, whereas NSUT13-289 and LK92-11 are considered to be sensitive to water stress. These results have significant implications for the development of new drought-tolerant sugarcane varieties. In conclusion, the research highlights the physiological and biochemical adaptations of sugarcane plants at various growth stages under water stress conditions. The findings suggest that enhancing the cultivation of stress-tolerant plants can be achieved through genetic improvement strategies, including biotechnological approaches or conventional breeding techniques. These approaches have the potential to contribute to the development of more resilient and drought-tolerant sugarcane varieties, which can enhance crop productivity and sustainability in water-limited environments.
82 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Figure 1 Gene expression of ProT in sugarcane drought stress region. qRT-PCR analysis of key Sugarcane genes related to drought: indicates significant changes of drought in Drought condition compared to control (t-test, p = 0.05). Figure 2 Gene expression of TPS in sugarcane drought stress region. qRT-PCR analysis of key Sugarcane genes related to drought: indicates significant changes of drought in Drought condition compared to control (t-test, p = 0.05). Figure 3 Gene expression of PST3 in sugarcane drought stress region. qRT-PCR analysis of key Sugarcane genes related to drought: indicates significant changes of drought in Drought condition compared to control (t-test, p = 0.05). Figure 4 Proline estimation of five sugarcane Figure 5 Trehalose estimation of five genotypes genotypes sugarcane under control conditions. under control conditions.
83 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Figure 6 MDA estimation of five sugarcane Figure 7 Hydrogen peroxide estimation of five under control conditions. sugarcane under control conditions. 6. REFFERENCE [1] Khumla, N., Sakuanrungsirikul, S., Punpee, P., Hamarn, T., Chaisan, T., Soulard, L. & Songsri, P. (2022). Sugarcane Breeding, Germplasm Development and Supporting Genetics Research in Thailand. Sugar Tech, 24(1), 193-209. [2] Lakshmanan, P. and Robinson, N. (2014). Stress physiology: Abiotic stresses, in Sugarcane: Physiology, Biochemistry, and Functional Biology, ed Moore P. H., Botha F. C. (Chichester: John Wiley & Sons, Inc.), 411–434. [3] Gentile, A., Dias, L. I., Mattos, R. S., Ferreira, T. H. and Menossi, M. (2015). MicroRNAs and drought responses in sugarcane. Front. Plant Sci. 6:58. 10.3389/fpls.2015.00058. [4] Ferreira, T. H., M. S. Tsunada, D. Bassi, P. Araújo, L. Mattiello,G. V. Guidelli, G. L. Righetto,V. R. Gonçalves, P. Lakshmanan and M. Menossi. (2017). Sugarcane water stress tolerance mechanisms and its implications on developing biotechnology solutions. Front. Plant Sci.8: 1077. [5] Yancey, P. H. (2001). Water stress, osmolytes and proteins. Am. Zool. 41,699-709. [6] Rathinasabapathi B. (2000) Metabolic engineering for stress tolerance: installing osmoprotectant synthesis pathways. Ann Bot. 86:709–716. [7] Gupta, R.A., Shah, N., Wang, K.C., Kim, J., Horlings, H.M., Wong, D.J., Tsai, M.-C., Hung, T., Argani, P., Rinn, J.L.(2010). Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076. [8] Iskandar, T. M., Rahmat, M. M., Noor, N. M., &Saleh, N. M. (2011). Corporate governanceand going concern problems: evidence from Malaysia. International Journal ofCorporate Governance, 2(2), 119-139. [9] Silva, E. T.; Ribeiro Filho, O. P. and Feio, R. N. (2011). Predation of native anurans by invasive bullfrogs in Southeastern Brazil: Spatial variation and effect of microhabitat use by prey. South American Journal of Herpetology 6(1):1-10. [10] Rasheed, M.I., Aslam, H.D. and Sarwar, S. (2010). Motivational Issues for Teachers in Higher Education: A Critical Case of IUB, Journal of Management Research,2(2):1- [11] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using Realtime quantitative PCR and the 2-ΔΔCt method. Methods 25: 402-408.23. [12] Dillewijn, C. van. (1952). Botany of Sugarcane. Chronica Botanica,Walthham, MA, USA. [13] Neves, M.J., Hohmann, S., Bell, W., Dumortier, F., Luyten, K., Ramos, J. , et al. (1995). Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis. Curr Genet 27: 110–122 [14] Queiroz, R. J. B., Santos, D. M., M., Carlin, S. D., Marin, A., Banzatto, D. A. and Cazetta, J. O. (2008) Osmoprotetores em cana-de-açúcar sob o efeito da disponibilidade hídrica no solo. Científica, v.36, p.107-115. [15] Beauchamp, C.O. and Fridovich, I.(1973).Isozymes of superoxide dismutase from wheat germ. Biochim. Biophys. Acta 317, 50–64. [16] Chance, B., and Maehly, A. C. (1955). Assay of catalase and peroxidases. Methods [17] Nakano, Y, and Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867-880.Enzymology.2:764-775. [18] Bradford M.M. (1976). A rapid sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-Dye Binding. Anal Biochem. 72:248–254. [19] Heath RL, Packer L. (1968). Photoperoxidation in isolated chloroplasts. I.Kinetics and stoichiometry of fatty acid peroxidation. Archives in Biochemistry and Biophysics.125:189–198. [20] Begum, K., and Islam, O.M. (2015). Selection of salt tolerant somaclones for development of salt stress tolerant varieties. Plant. 3 (4), 37-46.
84 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE [21] Hussain, A., Khan, Z. I., Ashraf, M., Rashid, M. H., and Akhtar, M. S. (2004). Effect of salt stress on some growth attributes of sugarcane cultivars CP-77-400 and COJ84. Int J Agric Biol. 6, 188-191. [22] Torres-Schumann, S., Godoy, J. A., Pintor-Toro, J. A., Moreno, F. J., Rodrigo, R. M., and Garcia-Herdugo, G. (1998). NaCl effects on tomato seed germination, cell activity and ion allocation. Journal of Plant Physiology. 135(2), 228-232. [23] Shannon, M. C. (1997). Adaptation of plants to salinity. Advances in Agronomy,60,75- 120.
85 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Nattapat Khumla1 *, Siwilai Lapbanjob¹, Wanlipa Suchato¹, Piyathida Insuk¹, Ratchada Pratchareanvanich², Supaporn Sukto³, Udomsak Duanmeesuk³, and Raweewan Chuekittisak6 1 Nakhon Sawan Field Crops Research Center, Nakhon Sawan, 60190, Thailand 2 Suphanburi Field Crops Research Centre, Suphanburi, 72160, Thailand 3Nakhon Ratchasima Agricultural Research and Development Center, Nakhon Ratchasima 30340, Thailand 4Uthaithani Agricultural Research and Development Center, Uthaithani, 61110, Thailand 5Ratchaburi Agricultural Research and Development Center, Ratchaburi 70120 Thailand 6 Field and Renewable Energy Crops Research Institute, Bangkok, 10900, Thailand *Correspondence to: Department of Agriculture, Nakhon Sawan Field Crops Research Center, Tak Fa, Nakhon Sawan, 60190, Thailand. [email protected] ABSTRACT: Sugarcane, valuable cash crop, plays a significant role in Thailand's economy. Due to the massive demand of high yielding sugarcane varieties in Thailand, Nakhon Sawan Field Crops Research Center (NSFCRC) of Department of Agriculture (DOA) developed a new sugarcane variety, DOA Nakhon Sawan1, through a breeding program. The developed variety is derived from a cross between Q76 and CP63-588 in 2010 which underwent five selection steps between 2011 and 2021. The efforts were focused on improving cane and sugar yields, good ratooning capacity, and adaptation to loam, clay-loam, and clay soils in various regions. The performance of DOA Nakhon Sawan1 was evaluated at eight regional locations with one plant and two ratoon crops. The developed variety performed well on loam, loam-clay, and clay-loam soils. In quality assessment, the commercial cane sugar (CCS) was 15.77, which is 10% and 7% higher than two widely grown varieties, LK92-11 and Khon Kaen3 (KK3), respectively, while sugar yield was 17.6 t/ha, 18% higher than LK92-11 and comparable to KK3. DOA Nakhon Sawan1 exhibited moderate resistance to red rot (Colletotrichum falcatum) and wilt (Fusarium moniliforme) but moderate susceptibility to smut (Ustilago scitaminea) based on artificial inoculation. Harvesting of DOA Nakhon Sawan1 in the early to mid-crushing season is recommended. The newly developed variety DOA Nakhon Sawan1, released for general cultivation on July 12, 2022, is expected to enhance the profitability of farmers, particularly in the central and northern regions of the country and minimize the production cost. Overall, DOA Nakhon Sawan1 is a promising new variety that offers high sugar yield and good adaptability, making it a valuable contribution to Thailand's sugarcane industry and related sectors. Keyword: Sugarcane, Sugarcane Breeding, High sugar, CCS, Cane yield. P-004 Exploring “DOA Nakhon Sawan1” a High Sugar-Yielding Sugarcane Variety and its Potential to Meet Growing Demand
86 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION The sugarcane industry is a vital component of Thailand's economic growth, contributing significantly to the country's agricultural production by serving as a raw material for various products such as sugar, ethanol, biomethane, biogas, and electricity. Moreover, sugarcane is a valuable contributor to environmental sustainability and generates employment and income for millions of people in Thailand. According to the sugarcane production data reported by Office of the Cane and Sugar Board (OCSB) in the 2022/23 crop year, the crop occupies approximately 7% of Thailand's agricultural area, covering 47 provinces in the North-Eastern, North, Central, and East regions, with 43%, 24%, 27%, and 6%, respectively [5]. The development of improved varieties has significantly impacted sugarcane production. Presently, Khon Kaen3 (KK3) variety released by Department of Agriculture (DOA) is the most notable variety [2] and due to high yield, good performances, and resilience characteristics occupied about 90% of sugarcane area in Thailand followed by LK92-11 (5%). However, in the last one decade, the average production of sugarcane in Thailand is quite low (60 t/ha) as compared to Brazil, India, and Australia [10]. Moreover, sugarcane production experienced a decline in certain years due to the recurring drought crisis, particularly in rainfed cane regions. The soil composition in the northeastern region primarily consists of sandy soil with low fertility, which adversely affects the crop's yield potential and diminishes its ability to regenerate through ratooning. In contrast, the northern and central areas are dominantly composed of loam, clay-loam, and clay soils, which have lower annual precipitation due to climate change. The long period of water scarcity can lead to a decrease in sugarcane production, and the presence of alkaline soils in some areas hinders the optimal functioning of essential micronutrients. Consequently, the growth and root system of sugarcane are impeded. As a result, this leads to reduced productivity in terms of cane and sugar content, as well as decreased viability for subsequent ratoon crops. Furthermore, the utilization of unsuitable cane varieties in these areas, along with the extensive cultivation of KK3, has raised concerns regarding genetic vulnerability and susceptibility to infectious diseases, which will have high potential impact on the sugarcane industry. This fluctuation in sugarcane production has resulted in a shortage of raw material supplies. Meanwhile, numerous sugar mills have expanded their crushing capacity and necessitated larger quantities of sugarcane to sustain the production of sugar and its related by-products. In response to reducing risks and increasing resilience,sugarcane breeding institutionsin Thailand are attempting to develop new varietiesfor distinct geographical locations, thereby enhancing sugarcane productivity.These breeding programs have been implemented in each region, encompassing the key cane-growing areas. Notably, Nakhon Sawan Field Crops Research Center (NSFCRC) played an important role in achieving substantial improvementsin sugarcane yields within rainfed regions characterized by loam, clay-loam, and clay soils. This program was conducted in 2008 to attain consistently high yields of both cane and sugar while prioritizing agronomic traits that are desirable for growers. It is anticipated that the introduction of these novel varieties will contribute to the enhancement of yields on loam, clay-loam, and clay soils. 2. METHODS DOA Nakhon Sawan1 is a sugarcane (a complex hybrid of Saccharum spp.) variety (former designation is NSUT10-266) developed by NSFCRC of DOA. The sugarcane breeding and selection process involved the general procedures of sugarcane breeding programs over the years in Thailand as follows [4]: Crossing The DOA Nakhon Sawan1 was derived from the cross Q76 × CP63-588 (Figure 1), conducted at the Suphanburi Field Crops Research Center (SPFCRC), Suphanburi province, Thailand in November 2010. The F1 seed of this cross and all seedlings of the 2010 series, were sown in January 2011 and germinated in a greenhouse of SPFCRC. Seedlings were transplanted into the trays. Each cross was labeled with its corresponding cross number. Figure 1 Pedigree chart of DOA Nakhon Sawan1 sugarcane variety
87 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Seedling stage (The first selection) The seedling stage began in April 2011 at NSFCRC, Nakhon Sawan province, Thailand. 10,933 F1 seedlings were transplanted into the field for the first selection using LK92-11 and KK3 as reference varieties. Each cross was planted with an 8 m row length, 1.5 m row spacing, and 0.5 m between plants. The number of rows for each cross depended on the number of F1 seedlings. Selected clones were harvested and designated individually in plant cane as NSUT10 series when the selected clones were developed by NSFCRC and bred by SPFCRC, which is located in U-Thong district. Selection in seedlings was visual and emphasized progeny yield, plant height, number of stalks per stool, stalk diameter, self-trashing, pith, amount of hairs (group 57) on the leaf sheath, and the occurrence of sugarcane diseases such as white leaf, red rot wilt, and smut diseases. Clonal stage (The second selection) In the following year, the selected clones, including DOA Nakhon Sawan1 from the first selection, were advanced to the second selection and planted in a single 8 m row length, 1.5 m row spacing, and 0.5 m between plants with double sugarcane seed setts (two-bud sett). The planting density was about 32 setts per row. 393 genotypes were arranged in an augmented design with three standard varieties, KK3, LK92-11, and K88-92, at a single site, NSFCRC, in February 2012–2013. Plant cane and the first ratoon crops from each plot were evaluated using cane yield performances and the same criteria described in the first selection, except for the measurement of CCS using a sample of six randomly selected stalks. Yield evaluation Preliminary trial (PT): After the second selection, the first trial was established at NSFCRC in late December 2013-2015. A total of thirty selected clones including DOA Nakhon Sawan1 were advanced to PT and arranged in a randomized complete block design (RCBD) with four replications conducted using KK3 and LK92-11 as reference varieties. Each clone was planted in four rows per plot in 8 m length, and 1.5 m row spacing. Both plant cane and the first ratoon crops were harvested at the plant age of 11-12 months. For each crop, the middle two rows were used for data collection i.e., yield components, millable stalks, stalk weight, stalk diameter, plant height, Brix (hand refractometer), and disease occurrence. Ten stalks for each of the four replications were sampled and analyzed for Commercial Cane Sugar (CCS) in SPFCRC. The sugar yield was calculated using the following equations: Sugar yield (t/ha) = Cane yield (t/ha) × (CCS/100) Standard trial (ST): DOA Nakhon Sawan1 and twelve selected clones with high cane yield, and sugar content from PT were further advanced to ST and evaluated. This stage involved evaluating the performance of the clones in three different locations i.e., NSFCRC, SPFCRC, and Sukhothai Agricultural Research and Development Center (SKARDC), Sukhothai province, Thailand in 2014-2016. At each location, the experimental design, plot size, reference varieties, and data collection were the same as described in PT. For the three consecutive years in plant cane, the first and second ratoon crops were harvested at the plant age 11-12 months. Farm trial (FT): In the subsequent period, from 2016 to 2020, the three clones that succeeded in the standard trial were subjected to FT. This stage involved evaluating the clones in eight locations of farmers’ fields across different sugarcane areas in Thailand: two locations in each of Nakhon Sawan and Kanchanaburi provinces and one location in Suphanburi, Nakhon Ratchasima, Uthaithani, and Sukhothai provinces. At each location, the experimental design and reference varieties were the same as described in ST, but the plot size was extended to six rows per plot. In the four middle rows of each plot in plant cane, the first and second ratoon crops were harvested and measured. The response of the genotype to varying environments for these clones’ adaptability was determined on plant cane crop using the linear regression model considered on the stability parameters; regression coefficient (b), while the environment and genotype-environment interactions were indicated on deviation from regression (S2d) using the Eberhart and Russell model [1]. Disease Resistance Screening for disease resistance was conducted under artificial inoculation at NSFCRC in 2014-2015. For resistance to red rot and wilt caused by Colletotrichum falcatum and Fusarium moniliforme, DOA Nakhon Sawan1 and three reference varieties, LK92-11 and KK3 (resistant reference variety), and NSS08-52-4-2 (susceptible reference variety) were inoculated using wound plug method by insertion the mycelia plug into the third internode above ground at six months after planting. The infected tissue was observed by splitting sugarcane stalk lengthwise and the number of invaded internodes was recorded after harvest. Assessed varieties were categorized according to
88 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE the severity of stalk tissue affected using the rating scale modified from Kalaimani reaction scale [3]. For smut disease reactions, LK92-11 and Marcos were resistant and susceptible reference varieties, respectively. The test varieties were inoculated by soaking in 5x106 spore suspension of Ustilago scitaminea before planting. The number of infected stools and the number of whips were recorded and calculated in plant cane and the first ratoon crops. Based on the smut stool infection and severity, sugarcane varieties were categorized using the rating scale described by U-Wanich [9]. Statistical Analyses The differences among genotypes across years and locations for cane yield, sugar yield, CCS, plant height, stalk diameter, single stalk weight, millable stalks, and internode length in cane were compared using the DMRT test at P = 0.05. The statistical analyses were processed with Statistix v8 software [8]. 3. RESULTS AND DISCUSSION DOA Nakhon Sawan1 is the latest high-sugar variety developed by NSFCRC of DOA. It is improved for clay, loam, clay-loam, and clay soils which is covering major sugarcane areas in the north, central regions, and some areas in northeastern Thailand. Yield Performance The cane yield of DOA Nakhon Sawan1 (112 t/ha) was 6% higher than the reference cultivars LK92-11 (106 t/ha) but had less cane yield than KK3 (118 t/ha). DOA Nakhon Sawan 1 had CCS (15.77) higher than LK92-11 (14.35) and KK3 (14.72) at 10% and 7%, respectively. When compared to the two reference varieties, the CCS of DOA Nakhon Sawan1 was higher than LK92-11 and KK3 at all tested locations and crops. The high heritability in sucrose content of DOA Nakhon Sawan1 may be affected by Q76, the female parent. The female parent plays a significant role in the inheritance of sucrose content in terms of brix. The crosses involving high-sucrose female parents contributed a large proportion of high brix progenies irrespective of the sucrose content of the male parent [7]. The sugar yield of DOA Nakhon Sawan1 was 17.6 t/ha, 18% higher than LK92-11 (15.0 t/ha) and comparable to KK3 (Table 1). Table 1. Yield performance of DOA Nakhon Sawan1 a Means calculated from 9 trials in plant cane of PT, ST, and FT trials b Means calculated from 7 trials in the first ratoon crop of PT, ST, and FT trials c Means calculated from 5 trials in the second ratoon crop of ST, and FT trials Agronomic traits The average of five agronomic traits (plant height, stalk diameter, single stalk weight, millable stalks, and internode length) of DOA Nakhon Sawan1 and reference varieties LK92-11 and KK3 calculated from PT, ST, and FT trials are listed in Table 2. The plant height of DOA Nakhon Sawan1 was 303 cm, not significantly different from LK92-11 (263 cm) or KK3 (284 cm). The stalk diameter of DOA Nakhon Sawan 1 (2.78 cm) was not significantly different from LK92-11 (2.73 cm) and KK3 (2.83 cm). DOA Nakhon Sawan1 (75,839 stalk/ha) had lower millable stalks than LK92-11 (85,658 stalk/ha) and KK3 (77,475 stalk/ha). The single stalk weight of DOA Nakhon Sawan1
89 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE (1.53 kg) was not significantly different from KK3 (1.60 kg) and greater than LK92-11 (1.37 kg). DOA Nakhon Sawan1 (10.8 cm) was longer than LK92-11 (9.40 cm) but not significantly different from KK3 (10.2 cm). Table 2. Agronomic traits of DOA Nakhon Sawan1 a Trait means calculated from PT, ST, and FT trials Botanical traits The botanical traits of DOA Nakhon Sawan1 were recorded and characterized from approximately 8-10 month-old plants following the descriptors described by Plant Varieties Protection [6], with LK92-11 and KK3. DOA Nakhon Sawan1 exhibited a cylindrical internode shape and had exposed internodes that were greenish-purple. There were no growth cracks on DOA Nakhon Sawan1 and two reference varieties. Various traits from different parts of the sugarcane plant were characterized in Table 3. Table 3. Botanical traits of sugarcane variety DOA Nakhon Sawan1 and reference varieties
90 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Adaptability The average performance, regression (bi), and squared deviation (S2di) for cane and sugar yields were shown in Table 4. DOANakhon Sawan1 exhibited high means, a regression coefficient value close to one, and a non-significant squared deviation. When evaluating yield performance, DOA Nakhon Sawan1 demonstrated promise and consistent results, indicating wide-range adaptability. Table 4. Stability analysis for yield performances of DOA Nakhon Sawan1. a Mean yield performance of FT, 6 locations in plant cane. Means in the same column followed by the same letter are not significantly different based on the DMRT test at P =0.05. b Stability parameters; b = Regression coefficient, S²d = Deviation from regression, ns = Non-significant, * = Significant at P=0.05 Disease Resistance Under artificial inoculation conditions, for red rot and wilt diseases, the invaded internode number of DOA Nakhon Sawan1, LK92-11, KK3, and NSS08-52-4-2 were 3.38, 1.39, 2.37, and 13.69 and categorized as moderately resistant (MR), resistant (R), moderately resistant (MR), and highly susceptible (HS), respectively (Table 5). For smut resistance, DOA Nakhon Sawan1 was 24.6%, rated moderately susceptible (MS), while LK92-11 and Marcos were 11.6% and 39.8%, rated as MR, and MS, respectively. Table 5. Red rot wilt and Smut resistance in sugarcane variety DOA Nakhon Sawan1 a R, resistant (1-2); MR, moderately resistant (2.1-4.0); HS, highly susceptible (>8). b MR, moderately resistant (7-20.00%); MS, moderately susceptible (21-60%). The completed data summaries were presented and approved by the Plant Breeding Research Committee of the DOA. DOA Nakhon Sawan1 was registered and released on July 12, 2022. 4. CONCLUSIONS The sugarcane variety, DOANakhon Sawan1 is now an alternative variety for cane growers cultivating sugarcane in loam, clay-loam, and clay soils, especially in Nakhon Sawan, Chainat, Suphanburi, and Nakhon Ratchasima provinces. The high sugar content will allow cane growers to earn more income per ton of cane. The cane is MS to smut disease, infectious planting areas should be avoided. Its harvesting in the early-mid crushing season is recommended. 5. ACKNOWLEDGEMENTS The breeding of DOA Nakhon Sawan1 was funded by the National Research Council (NRCT) of Thailand, the Thailand Science Research and Innovation (TSRI), and the Department of Agriculture (DOA) is fully acknowledged by the authors. 6. REFERENCES [1] Eberhart, S. A., and Russell, W. A. 1966. Stability parameters for comparing varieties. Crop Science, 6, pp. 36-40. [2] Jaisil, P., P. Songsri, N. Jongrangklang, J. Kruangpathi, and K.Suttapakdee. (2020). National evaluation of elite sugarcane clones suitable for growing areas Phase III (Period II), Final report. Khon Kaen Univeristy. (in Thai)
91 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE [3] Kalaimani, T. (2000). Pathogenic variability of red rot caused by Colletotrichum falcatum Went. In Tamil Nadu, Indian sugar. pp.841-846. [4] Khumla, N., S. Sakuanrungsirikul, P. Punpee, T. Hamarn, T. Chaisan, L. Soulard, and P. Songsri. (2022). Sugarcane Breeding, Germplasm Development and Supporting Genetics. Sugar Tech. pp. 193–209, DOI: 10.1007/s12355-021-00996-2 [5] Office of The Cane and Sugar Board. (2023). Sugarcane plantation areas in Thailand crop year 2022/23 report. Office of The Cane and Sugar Board, Ministry of Industry. https://www.ocsb.go.th/2023/reports-articles/ area-yield/21623/. (in Thai). (accessed April 2023) [6] Plant Varieties Protection Division. (2005). Manual of plant variety characteristic examination: Sugarcane. Plant Varieties Protection Division, Department of Agriculture. http://lib.doa.go.th/multim/e-book/EB00147. pdf. (in Thai). (accessed April 2023) [7] Shanthi, R.M., Alarmelu, S. & Balakrishnan, R. (2005). Role of female parent in the inheritance of brix in early selection stages of sugarcane. Sugar Tech 7, pp. 39–43. https://doi.org/10.1007/BF02942527 [8] Statistix. (2015). Statistix analytical software. v8. http://www.statistix.com/ (accessed Jan 2015). [9] U-Wanich, W., Srisink, S., and Sakulwong, A. (1991). Study on sugarcane smut disease. In Proceedings of the 29th Kasetsart Annual Conference, pp 505-513. February 4-7, 1991, Bangkok, Thailand. (in Thai). [10] Yadav, S., P. Jackson, X. Wei, EM. Ross, K. Aitken, E. Deomano Atkin, BJ. Hayes, and KP. Voss-Fels. (2020). Accelerating gain in sugarcane breeding using genomic selection. Agronomy 10: 1–21. https://doi.org/10.3390/ agronomy10040585.
92 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Uraiwan Pongpayaklers1 *, Piyatida Insuk1 and Suwat Phoonphan1 ¹Department of Agriculture, Field Crops and Energy Renewable Crops Research Institute, Suphan Buri Field Crops Research Center, Suphan Buri Province, 72160, Thailand *Correspondence to: Department of Agriculture, Field Crops and Energy Renewable Crops Research Institute, Suphan Buri Field Crops Research Center, Chorakhesamphan Subdistrict, U-Thong District, Suphan Buri Province, 72160, Thailand. [email protected] ABSTRACT: Study on smut disease reaction of the promising sugarcane clones series 2015 was conducted at Suphan Buri Field Crops Research Center. Twelve clones of the promising sugarcane clones were planted and com pared with LK92-11 (Resistant check), Marcos (Susceptible check) and KK3. They were dipped in spore suspension of Sporisorium scitamineum (Syd.) which is a causal agent of sugarcane smut. After that they were incubated overnight before planting.Incidences ofsugarcane smut were counted every month until the canes were 10 months old and harvested at 12 months to evaluate the ratoon reaction. In plant canes, all of the tested canes were moderately susceptible (MS) reaction to susceptible (S) reaction where LK92-11, UT15-060 and UT15-094 had the MS reaction while other clones/cultivars had S reaction. UT10-023 gave the highest cane yield (176.3 tons/ hectare). In ratoon canes, there was only 1 clone, UT15-094 had the moderate resistant (MR) reaction while KK3 and LK92-11 had MS reaction and the other clones/cultivars had S reaction program. KK3 gave the highest cane yield (165.0 tons/ hectare) but was not significantly different from UT15-299 (147.5 tons/ hectare). The Smut disease reaction of the promising sugarcane clones Series 2015 will be considered before entering the farm trial (FT) for further breeding Keyword: Sugarcane disease, Smut disease, Sporisorium scitamineum, Ustilago scitaminea. P-005 Smut Disease Reaction of the Promising Sugarcane Clones Series 2015 under Irrigated Condition
93 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE 1. INTRODUCTION Sugarcane smut is caused by Sporisorium scitamineum (Syd.) [1] (Synonyms: Ustilago scitaminea Syd). The first outbreak and damage to sugarcane cultivation in Thailand was found in 1964 [2]. Currently, smut can be found everywhere in sugarcane cultivation, and it is an increasing trend to epidemic. Symptoms of the disease can be identified by a long black whip-like structure from the growing point of the canes instead of the normal shoot. The structure may be straight or curl and the length ranges about 1 – 2 cm. to 150 cm. [3]. Infected sugarcane will stop growing and split a lot. If the symptoms are severe, the cane will be stunted, thin, and the internodes will be very short. Cane does not develop into stalk and dies. As a result, the yield and quality of sugarcane are reduced by 50-80% and the ability for ratooning is reduced. Yield losses vary according to the disease resistance level of each sugarcane variety which will cause the severity of the disease to be different [4]. If the sugarcane is infected when the age is over 7 months, the yield will not be different from the normal sugarcane [5]. It has been reported that sugarcane severely infected with smut effect to sugarcane yield reductions up to 3.85 tons/hectare [6]. The fungal spores attached to the seed can be blown away by the wind. They do not only survive in the soil for long periods of time, but also spread disease to sugarcane new crop. Prevention and elimination of smut disease can be done in many methods. One effective method is to treat sugarcane seed with hot water. Treatment at 50°C for 2 hours or at 52°C for 30 minutes before planting can reduce the incidence of disease up to 75% [7]. In addition, using disease-resistant varieties are also an alternative method to prevent or reduce the spread of disease. Therefore, the development of new sugarcane varieties to have potential for yield and resistant to diseases must be important as well. The purpose of this experiment was to test the smut disease reaction of the promising sugarcane clones Series 2015 under irrigated condition for selection in breeding program before entering the farm trial (FT). It is useful information for certification and suggestion to farmers in the future. 2. MATERIALS AND METHODS Materials 1. High-yielding sugarcane clones include 7 clones of series 2015, 2 clones of series 2010 and 3 compared varieties namely LK92-11 (Resistant check), Marcos (Susceptible check), and KK3, total 12 clones/varieties aged 8-10 months. 2. Spores of Sporisorium. Scitamineum Methods Experimental design is Randomized Completely Block Design (RCBD), 3 replications, planting 3 rows/ replication. Conducted at Suphanburi Field Crops Research Center (SFCRC). 1. Prepared spore suspension of S. scitamineum 5x106 spores/ml 2. Prepared sugarcane seeds with 2 buds/ sugarcane seed and soaked in the spore suspension of S. scitamineum for 30 minutes. The sugarcane seeds were incubated overnight. 3. The inoculated sugarcane seeds were planted the next day. The spacing was 0.5 x1.5 m, 2 sugarcane seeds/ hole. Applying 15-15-15 chemical fertilizer at the rate of 312 kg/ hectare, divided into 2 times, planting time and when the sugarcanes were 2.5 months old, pesticides were used as recommended by the Department of Agriculture. 4. Recorded germination data at 45 days and disease evaluation [8] every 1 month to 10 months. 5. Sugarcanes were harvested at 12 months of age and maintained ratoon 1st canes. 6. Data were analyzed-Analysis of Variance (F-test) and compared with means by DMRT method. 7. The ratoon 1st canes were maintained and recorded data in the same way as plant canes. RATING SYSTEM
94 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Severity score Level 1: There are 1-2 whips, normal growth. Level 2: There are 2-3 whips, reduced growth, more tillering, small stalks. Level 3: There are 3-4 whips, stunted, more tillering, mostly small and frayed. Level 4: There are more than 4 whips, clumps like lemongrass, no yield and dies. 3. RESULTS AND DISCUSSION In plant canes, the results showed that the percentage of germination was between 97.2 - 100%. The 2015 series, UT15-060, had the lowest percentage of disease stool 46.3%, followed by clone UT15-094 with 50.0% of disease stool. Both clones had moderately susceptible (MS) reaction and a severity score was 3, while the LK92-11 (Resistant check) reaction had 45.4% of diseased stool, a reaction to MS and a severity score was 3. Marcos (Susceptible check) and KK3 had 61.1% and 55.1% of diseased stool respectively, a reaction to susceptible (S) and the severity score was 4. The other clones tested had 63.2 - 94.3% of the diseased stool, a reaction to S and the severity score were 4. UT10-023 gave the highest sugarcane yield with 176.3 tons/ hectare which was not significantly different from UT15-299 with 140.6 tons/ hectare, while LK92-11 and KK3 had the same yield of 119.4 tons/hectare. UT15-060 had the highest number of stalks with 93,825 stalks/ hectare which was not significantly different from UT10-023, UT14-034, UT15-147, UT15-267, UT15-299, Marcos and LK92-11 (Table.1). In ratoon canes, the percentage of germination was between 76.85 - 99.07%. UT15-094 had the lowest percentage of disease stool 14.2%, had moderately resistant (MR) reaction and a severity score was 1, while the LK92-11 and KK3 had 34.8% and 60.2% of disease stool respectively. Both varieties had the MS reaction and a severity score were 1 and 3 respectively. KK3 gave the highest sugarcane yield 165.0 tons/hectare but it was not significantly different from UT15-299 147.5 tons/hectare. UT15-299 had the highest number of stalks 107,088 stalks/hectare which was not different from KK3 UT15-060, LK92-11, UT15-034 and UT10-023. (Table 2). UT15-094 had the lowest percentage of disease stools in both plant cane and ratoon cane (Figure 1). In the plant cane, the percentage of disease stool was 50.0%, a reaction was MS and a severity was 3. In ratoon cane, the percentage of disease stool decreased by 14.2%, the reaction was MR, the severity was 1 and the yield was normal. The yield and yield components did not vary inversely with disease level. The yield of sugarcane also depended on the potential of each clones/varieties. Although there were high percentages of diseased stool in some clones/ varieties, they still gave yield normally according to the potential of the variety. For example, UT10-023 had 63.2% disease stool in plant cane and 94.6% diseased stool in ratoon cane, but there were S reaction with a severity score of 4 and gave the sugarcane yield of 176.3 tons/hectare in plant cane and 118.8 tons/hectare in ratoon cane which was not different from sugarcane grown under normal conditions (non-inoculated). It has been reported that UT10-023 gave the cane yield of 170 tons/ hectare in plant cane and 108.8 tons/ hectare in ratoon cane [9]. It showed smut disease tolerance same as UT15-094 and UT15-229 while there were high percentages of the disease stool and yield decreased in some clones/varieties. Such as UT15-337 which had 94.3% and 100% of disease stool in plant cane and ratoon cane respectively. There was S reaction and severity score 4 and has yields of 85 and 51.3 tons/hectare respectively. The yield reduced from growing in normal conditions that were not inoculated. (135.6 tons/hectare in plant cane and 105 tons/hectare in ratoon cane [9]). This clone showed susceptibility to smut disease. Therefore, it should not be selected for further planting. Figure 1 Percentage of disease stool in plant cane and ratoon 1st
95 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Table 1 Reaction of smut disease in sugarcane and yield UT series 2015: plant cane ns, ** = non significant, significant at P ≤ 0.01. Means followed by the same letter in the same column are not significantly different at the 1% level by DMRT. Table 2 Reaction of smut disease in sugarcane and yield UT series 2015: ratoon1st ns, ** = non significant, significant at P ≤ 0.01. Means followed by the same letter in the same column are not significantly different at the 1% level by DMRT. 4. CONCLUSIONS UT15-094 had the lowest percentage of diseased stool in plant cane and ratoon cane. UT10-023, UT15-094 and UT15-229 were tolerated to smut disease due to S reactions but they gave yields normally. UT15-337 was susceptible to smut disease due to S reactions and yield was greatly reduced when smut disease infected. 5. ACKNOWLEDGEMENT Thank you, Thailand Science Research and Innovation (TSRI) for research funding Suphan Buri Field Crops Research Center (SFCRC), Field and Renewable Energy Crops Research Institute (FCRI) , Department ofAgriculture (DOA) for all supports. 6. REFERENCES [1] Piepenbr, M., Stoll, M. and Oberwinkler, F. (2002). The generic position of Ustilago maydis, Ustilago scitaminea, and Ustilago esculenta (Ustilaginales), Mycological Progress, Vol.1, No. 1, pp. 71–80. [2] Martin, J.P.(1964). A survey of sugarcane disease in Thailand. Bangkok Sugar. Indust. 28 p. [3] Jack, C.C., Stephen A.F. and Thomas L.T. (1983). Hawaii’s Approach to control of sugarcane smut. Plant Dis. 67 pp. 452-457.
96 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE [4] Uwanich, W., Srisingh, S. and Kusonwong, A. (1985). evaluation of damage to sugarcane yield due to smut disease. Research Report 1985. Division of Plant Pathology and Microbiology. Department of Agriculture. pp. 1446-1456. [5] Uwanich, W. (2002). Important sugarcane diseases caused by fungi. Agronomy Research Group, Division of Plant Pathology and Microbiology, Department of Agriculture, Ministry of Agriculture and Cooperatives. [6] Glaz, B., Ulloa M.F. and Parrado R. (1989). Yield effects of sugarcane smut infection in Florida. American Society of Sugar Cane Technologists. Vol 9 pp. 71-80. [7] Srising, S., Uwanich, W., Kusolwong, A. and Chairin, S. (1985). Effects of hot water soaking method for eradicat major diseases on sugarcane seed with sugarcane varieties that are popularly planted in Thailand. Research report 1985 Division of Plant Pathology and Microbiology, Department of Agriculture. pp. 1473-1485. [8] Uwanich, W., Srising, S., Kusonwong, A. (1991). Study of sugarcane smut disease. Kasetsart University conference, No. 29, 4-7 February 1991. pp. 505-513. [9] Insook, P., Amornpol, V., Pantu, M., Nukaew, K., Suwanpong, S. (2021). Annual Research Report 2021. Suphanburi Field Crops Research Center. Department of Agriculture Ministry of Agriculture and Cooperatives. pp. 183-195.
97 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Wasana Wandee1 *, Piyatida Insuk1 , Somboon Wandee1 , Nattapat Khumla2 1 Department of Agriculture, Field and Renewable Energy Crops Institute, Suphaan Buri Field Crops Research Center, 72160, Thailand ²Department of Agriculture, Field and Renewable Energy Crops Institute, Nakhon Sawan Field Crops Research Center, 60190, Thailand *Correspondence to: Department of Agriculture, Field and Renewable Energy Crops Institute, Suphaan Buri Field Crops Research Center, 72160, Thailand, [email protected] ABSTRACT: Nitrogen use efficiency is a key factor to increase sugarcane yield. It is different in each sugarcane variety base on their genetic background, which will determine the optimal nitrogen fertilizer application for new sugarcane variety. The objective of the experiment was to study nitrogen use efficiency for nitrogen fertilization recommendation in a new sugarcane variety. The experiment was conducted in loam soil, Kamphaeng Saen soil series during February, 2022 - February, 2023. The experimental design was a split plot in RCBD with 4 replications. Main plot was comprised of 5 clones/varieties of sugarcane including 3 promising clones i.e. UT15-060 UT15-094 UT10-044, and 2 check varieties (Khon Kaen 3 and LK92-11). Sub plot was consisted of 5 nitrogen fertilizer rates i.e. 0, 7.5, 15, 22.5 and 30 kg N/rai. Phosphate and potassium fertilizers were applied base on soil analysis at the rate of 3 kg P2O5/rai and 6 kg K2O/rai. The yield was recorded and calculated nitrogen use efficiency (NUE). The result showed that there was no interaction between the main plot and the sub plot. Cane yield and sugar yield were affected by nitrogen fertilizer rate. For cane yield, nitrogen fertilizer at rates 7.5, 15, and 30 kg N/rai gave the cane yield 21.89, 22.52, and 22.33 ton/rai, respectively, which were higher than no nitrogen fertilizer application (19.56 ton/rai) but nitrogen fertilizer at a rate 22.5 kg N/rai was not significantly different when compared with no nitrogen fertilizer application. For sugar yield, nitrogen fertilizer at rates of 7.5, 15, and 30 kg N/rai gave the sugar yield of 2.95, 3.01, and 3.05 ton CCS/rai, which were higher than no nitrogen fertilizer application (2.66 ton CCS/rai), but nitrogen fertilizer at a rate 22.5 kg N/rai was not significantly different with no nitrogen fertilizer application. In terms of nitrogen use efficiency, Khon Kaen 3 variety had the highest nitrogen use efficiency (0.50 ton yield/kg N) at 7.5 kg N/rai nitrogen fertilizer followed by LK92-11, UT15-094 and UT15-060, which had nitrogen use efficiency 0.50, 0.35 and 0.21 ton yield/ kg N respectively, when applied nitrogen fertilizer at 7.5 kg N/rai. For clone UT10-044 had nitrogen use efficiency at 0.11 ton yield/kg N when applied nitrogen fertilizer at 7.5 and 15 kg N/rai. These results suggest that the optimal nitrogen application for Khon Kaen 3, LK92-11, UT15-094, and UT15-060 should be at a rate 7.5 kg N/rai, while UT10-044 should be at rates 7.5 and 15 kg N/rai, to high nitrogen use efficiency for sugarcane productivity. Keyword: sugarcane, loam soil, Kamphaeng Saen soil series, nitrogen use efficiency P-006 Nitrogen Use Efficiency of the Promising Sugarcane Clones Series 2015 under Irrigated Condition, Suphan Buri Province
98 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry AGRICULTURE & MACHINERY INCLUDES SMART FARM AND PRECISION AGRICULTURE Sangduan Chanachai1 *, Theerarat Chinnasaen1 , Piyarat Jangpol1 , Ammarawan Tippayawat¹, Werapon Ponragdee² and Weerayuth Srathongkan1 ¹Khon Kaen Field Crops Research Center, Khon Kaen, 40000, Thailand ²91/1 Moo.10, Natoong, Mueang Chumphon, Chumphon, 86000, Thailand *Correspondence to: Khon Kaen Field Crops Research Center, Address, Khon Kaen,40000, Thailand. E-mail [email protected] ABSTRACT: Sugarcane is the major sugar-producing crop worldwide. In Thailand, most of the sugarcane production areas are in sandy to loamy sand soil. The sugarcane selection suitable for the area is an important procedure to be considered in the early stage of sugarcane breeding to the release of a new variety. Therefore, the aim of this study was to hybridization and selection on sugarcane series 2018 for sandy to loamy sand soil in rainfed condition in Thailand. This experiment was conducted in 2017 to 2020 in two locations; (1) Tha Phra site, Khon Kaen Field Crop Research Center, Khon Kaen Province, and (2) Loei Horticulture Research Center, Loei Province, hybridization was bi-parental cross and poly cross. The 1st selection stage was conducted by family selection with 3 replications, the standard varieties as KK3, K88-92 and LK92-11, the aspect traits of this stage were good agricultural traits, high yield and yield components, after carried out in this stage 194 clones (87 combinations) were selected from 445 combinations with 17,561 seedlings. The experimental design in the 2nd selection stage was the Augmented Randomized Complete Block Design with the standard varieties as KK3, K88-92, LK92-11 and KK07-250, the aspect traits of this stage were good agricultural traits, high cane yield and yield component as sugar yield, sugar content or commercial cane sugar (CCS), 68 clones with 48 combinations were selected in this stage and continue to study in preliminary yield trial and other study of breeding program to obtain the new sugarcane varieties from promising clones series 2018 those expected to be good characteristics and contribute the sugarcane production in sandy to loamy sand soil in Thailand in the future. Keyword: Sugarcane, Selection, Hybrids, Promising Clones P-007 The Selection of Sugarcane Promising Clones Series 2018 for Sandy to Loamy Sand Soil in Thailand