PROCEEDINGS OF SUGARCANE 2023

99 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 Theerarat Chinnasaen1 *, Sangduan Chanachai¹, Chayan Pakdeethai¹, Ammarawan Tippayawat¹ and Krongkan Pongpanchamit¹ 1 Khon Kaen Field Crops Research Center, Khon Kaen, 40000, Thailand *Correspondence to: Khon Kaen Field Crops Research Center, Address, Khon Kaen, 40000, Thailand. E-mail: [email protected] ABSTRACT: The Northeastern is one of the most sugarcane production areas in Thailand, they have a unique environmental condition for sugarcane cultivation, hence the aims of this study were to evaluate (1) the photosynthetic parameters of sugarcane promising clones and (2) the correlation between those parameters to improve the new sugarcane varieties including to present the information of its in different plant age. This study was conducted in Khon Kaen Field Crops Research Center (KKFCRF), Khon Kaen Province, Thailand, during January to November 2022, three promising clones (KK11-516, KK14-030, and KK14-136) and two standard varieties (LK92-11 and KK3) were cultivated with 3 replications, measured the photosynthetic parameters at 4, 6, 8, and 10 months after planting (MAP), and evaluate total soluble solids (TSS) at 10 MAP. The results showed that the highest net photosynthesis rate (A) was KK14-136 as 20.24 µmolCO2m-2s-1 at 10 MAP, the stomatal conductance (gs) were tended to increased when plant age was raised, but under the effect of environment such as at maturity period (before sucrose accumulation in sugarcane) in the winter the photosynthetic parameters were lower than the previous developmental stage, meanwhile the water use efficiency (WUE) was not significant when compared between sugarcane varieties or plant age, and for the correlation coefficients obtained that the A was highly significant and rather high positive correlation with the other, excepted the TSS that not non-significant with the other parameter. This study can provide the photosynthetic parameters data of the sugarcanes under the Northeastern Thailand condition and indicated that the appropriate duration for photosynthetic evaluation can be either 8 or 10 MAP, especially in KK14-136 that presented high A and gs; however, to confirms these results, the data of yield and yield component are crucial to emphasize the recommended promising clones for future study. Keyword: Net photosynthesis rate, Transpiration rate, Stomatal conductance, Water use efficiency P-008 The Photosynthetic Parameters of Sugarcane Promising Clones in the Northeastern Thailand

100 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 Photosynthesis is the basis of yield formation. It consists of two stages. The first, called light reaction, refers to the reactions related to light capture, electron transport and the formation of reducing power (i.e. NADPH and ATP). The second is the carbon reduction cycle, which consumes ATP, NADPH, and produces carbohydrates. According to sugarcane is C4 plant, has the mechanism to improve the photosynthetic efficiency such as permits the concentration of CO2 in the vascular bundle to reach 10 times the atmospheric concentration within cell that contains Rubisco. However, the rate of photosynthesis in a plant is dependent on the environmental changes; availability of water and nutrients in soil, temperature, humidity, and light etc., as well as to intrinsic features of plant, such as the demand of photoassimilates and the expression of certain genes (Souza and Buckeridge, 2014; Santos and Diola, 2015). The Northeastern region (ISAN) of Thailand is the largest rainfed sugarcane cultivation area, there have the unique environmental conditions; high temperature, drought, and low or various of rainfall etc., those factors have an effect on sugarcane growing including the photosynthesis which supporting source of sugar in the yield, meanwhile KK3 is the most widely planted variety over the last decade (Buakom et al., 2020). As a result, it could be risk to get inbreeding depression of variety in case of the epidemics of plant diseases, insect pests, or low of agronomic traits, at the same time the knowledge of the photosynthetic parameter of regular sugarcane varieties that cultivated in ISAN were limited, furthermore the new sugarcane varieties could be the one of solutions to cope sugarcane cultivation in this area in the future. Therefore, the aims of this study were to evaluate (1) the photosynthetic parameters of sugarcane promising clones and (2) the correlation between those parameters to support the breeders collect the new sugarcane varieties. 2. MATERIALS AND METHODS Plant material and growth condition Two groups of sugarcane plants (Saccharum officinarum L.), the sugarcane promising clones; KK11-516, KK14-030, and KK14-136 which improved the variety by Khon Kaen Field Crops Research Center (KKFCRC) and (2) the standard varieties; LK92-11 and KK3, were grown in 80 cm diameter x 100 cm height of cement blocks with contained 345 kg of potting soil (from sugarcane field) per experimental unit, the chemical properties were 5.4 for pH, 0.033 dS/cm for EC, organic matter, total N, available P, exchangeable K, and exchangeable Ca as 0.32%, 0.014%, 27.49 mg/kg, 47.21 mg/kg, and 280.53 mg/kg, respectively. The irrigation was applied according to sugarcane water requirement (Paisancharoen et al., 2012) and applied chemical fertilizer grades 15-15-15 at rate of 312.5 kg/ha at planting date and 60 days after planting. The experiment was conducted during January to November, 2022 in KKFCRC (16º28’55.6”N and 102º 49’35.3”E), Sila, Mueang Khon Kaen, Khon Kaen Province, Thailand, under natural environmental conditions of the Northeastern Thailand. Meteorological data (Figure 1) were taken from the KKFCRC’s weather station that located about 1.5 km away from the experimental field, the data was in at the start to 10 months after planting (MAP), the range of minimum temperature, maximum temperature, relative humidity, and total rainfall were collected as 14.5-27.5 ºC, 21.5-39.0 ºC, 56-100%RH, and 1721.1 mm, respectively. Moreover, developmental stages of sugarcane and seasons in the Northeastern Thailand during sugarcane development were also presented in the Figure 1. Figure 1. Daily climate, developmental stages of sugarcane, and seasons during conducted the experiment at KKFCRC, Khon Kaen Province on 1 February to 30 November, 2022 or 0 to 10 MAP.

101 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 Experimental design and data collection The experimental design was the randomized completely block design (RCBD) by sugarcane varieties as the treatments, KK11-516, KK14-030, KK14-136, LK92-11, and KK3 with 3 replications, the photosynthetic parameters were the net photosynthesis rate (A), internal CO2 concentration (Ci), transpiration rate (E), and stomatal conductance (gs), were measured by the Lcpro TAdvanced Portable Photosynthesis System (ADC BioScientific Ltd., Hoddesdon, UK), the second or third youngest, fully expanded, healthy leaves of representative plants were selected in each plot or experimental unit. Measurements were made between 8:00 to 11:00 h on sunny days at 4, 6, 8, and 10 MAP, the chamber light source of LCpro T Advanced Portable Photosynthesis System was set at 1500 µmol m-2s-1 (Liu et al., 2020), the CO2 concentration and temperature were based on the ambient condition in each collection date. Calculated the water use efficiency (WUE) as the follow formula, WUE = A/E (Sun et al., 2011); moreover, to evaluate the correlation between the photosynthetic parameters and sugar accumulation in sugarcane, hence this study was measured the total soluble solids (TSS) of sugarcane in each experimental unit by collected the sugarcane juice at the middle of its stalks and evaluated the TSS with the digital refractometer (OPTi® Digital Handheld Refractometer, Bellingham + Stanley, UK) Statistical analysis The comparison between the mean of sugarcane varieties or collection duration time in all parameters were made by using the duncan multiple range test (DMRT’s test) at the 5% level. The coefficient of correlation of all parameters were analyzed by pearson correlation. 3. RESULTS AND DISCUSSION When compared with genotypes, the net photosynthesis rate at 10 MAP was significant, KK14-136 presented the highest as 20.24 µmolCO2m-2s-1 , when compared with plant age, at 8 MAP showed the highest as 23.08 µmolCO2m-2s-1 for KK11-516, and for KK3 at 6 MAP showed the highest as 21.34 µmolCO2m-2s-1 but non-significant when compared with the net photosynthesis rate at 8 MAP as 20.35 µmolCO2m-2s-1 . For the internal CO2 concentration was highly significant at 8 MAP when compared with genotypes, KK14-136 and KK14-030 were the highest as 228.17 and 199.50 µmolmol-1, respectively while the highest internal CO2 concentration tended to increse at 10 MAP for LK92-11 and KK3 as 197.17 and 212.17 µmolmol-1, respectively. For the transpiration rate, when compared with genotypes it was significant at 8 MAP, KK14-136 was the highest as 3.46 mmolm-2s-1, and when compared with plant age, the transpiration rate was high at 6 or 8 MAP for KK11-516 and KK3 with highly significant and significant, respectively. For the stomatal conductance, when compare with genotypesit wassignificant at 10 MAP, KK14-030 was the lowest as 0.12 mmolm-2s-1, meanwhile when compare with plant age, all genotypes presented high level at 8 MAP with the average as 0.20 mmolm-2s-1. For the water use efficiency was not significant when compared with genotypes or plant age. For TSS, KK3 showed the highest as 20.3 ºBrix with significant (Figure 1), and for the correlation coefficients obtained that the net photosynthesis rate was highly significant and rather high positive correlation with the other (E, gs, and WUE), excepted the TSS that not non-significant with the other parameter (Table 1).

102 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. Net photosynthesis rate (A), internal CO2 concentration (B), transpiration rate (C), stomatal conductance (D), water use efficiency (E) of various sugarcanes varieties at 4, 6, 8, and 10 months after planting, and total soluble solids at 10 months after planting (F), (data are mean±SE), ( ) for compared between genotypes, [ ] for compared between duration time, *=Significant at p < 0.05, **=Significant at p < 0.01. Table 1. Correlation coefficients among photosynthetic parameters and TSS of 5 genotypes at 10 months after 1/*Correlation is significant at the 0.05 level (2-tailed) **Correlation is significant at the 0.01 level (2-tailed) ns=not significant In this study, the photosynthesis parameters showed difference when compared with genotypes, similar to Luo et al. (2013) reported that the photosynthesis rate and stomatal conductance could be affected greatly by the specific combining ability among sugarcane varieties. Similar to Luo et al. (2014) reported that 17 sugarcane varieties were classified to four clusters base on the photosynthetic parameters especially at the early elongation stage of sugarcane growing; moreover, the photosynthesisrate had a positive correlation with transpiration rate and stomatal conductance as significant and highly significant correlation coefficient of 0.565 and 0.608 as well as transpiration rate has a positive correlation with stomatal conductance as significant correlation coefficient of 0.679 as a result Endres et al. (2010) also concluded that the photosynthetic activity of sugarcane depends greatly on stomatal conductance. In this study, the sugarcanes responded to their environments such as at maturity period in the winter that low temperature and humidity, the photosynthetic parameters tended to decreased. 4. CONCLUSION The photosynthetic parameters of sugarcane were differenct according to genotypes or plant age as well as the appropriate duration time for photosynthetic evaluation in sugarcane can be either 8 or 10 MAP, with KK14-136 presented good charecteristics of photosynthetic parameters especially in high the net photosynthesis rate or transpiration rate including stomatal conductance that probably related to the capacity of sucrose synthesis in the carbon reduction cyce, and connected to the accumulation of sugar in storage parenchyma cells (need more study). Moreover, the net photosynthesis rate was high positive correltion with transpiration rate, stomatal conductance, and water use efficiency that presented the relation of photosynthetic parameters in sugarcane. 5. ACKNOWLEDGMENT This work was funded by the Thailand Science Research and Innovation (TSRI).

103 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 6. REFERENCES Buakom, W., P. Krachal, S. Gonkhamdee, P. Songsri and N. Jongrungklang. 2020. Responses of rooting and physiological characteristics of sugarcanes grown under mimic drought stress as low water potential at early stage. Khon Kaen Agriculture Journal, 48(6): 1442-1457. Endres, L., J.V. Silva, V.M. Ferreira and G.V.S. Barbosa. 2010. Photosynthesis and water relations in brazilian sugarcane. The Open Agriculture Journal, 4: 31-37. Liu, Y.Y., J. Li, S.C. Liu, Q. Yu, X.J. Tong, T.T. Zhu, X.X. Gao and L.X. Yu. 2020. Sugarcane leaf photosynthetic light responses and their difference between varieties under high temperature stress. Photosynthetica, 58(4): 1009-1018. Luo, J., Y.B. Pan, L. Xu, Y. Zhang, H. Zhang, R. Chen and Y. Que. 2014. Photosynthetic and canopy characteristics of different varieties at the early elongation stage and their relationships with the cane yield in sugarcane. The Scientific World Journal, vol. 2014, Article ID 707095, 9 pages. Luo, J., Y.X. Que, H. Zhang and L.P.Xu. 2013. Seasonal variation of the canopy structure parameters and its correlation with yield related traits in sugarcane. The Scientific World Journal, vol. 2013, Article ID 801486, 10 pages. Paisancharoen, K., T. Sansayawichai, S. Luanmanee, S. Thippayarugs, K. Chusorn, J. Chuenrung and C. Pakdeethai. 2012. Water requirement and Kc values of Khon Kaen 3 sugarcane variety. Khon Kaen Agriculture Journal (Supplement), 3: 103-114. (in Thai) Santos, F. and V. Diola. 2015. Physiology. p.13-33. Santos, F., A. Borem, and C. Caldas (Eds). In Sugarcane Agricultural Production, Bioenergy, and Ethanal. Elsevier. Waltham. Souza, A.P. and M.S. Buckeridge. 2014. Photosynthesis in sugarcane and its strategic importance to face the global climatic change. p.359-364. In Luis Augusto Barbosa Cortez (Coord.). Sugarcane bioethanol-R&D for Productivity and Sustainability, São Paulo: Editora Edgard Blücher, 2014. Sun, J.K., T. Li, J.B. Xia, J.Y. Tian, Z.H. Lu and R.T. Wang. 2011. Influence of salt stress on ecophysiological parameters of Periploca sepium Bunge. Plant, Soil and Environment, 57(4): 139–144.

104 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 Chartchai Chotisan1 *, Thanyamas Buatkrathok¹, Supichaya Mongpraneet¹, Sawai Buddiwong² and Wanthanan Sinchai² ¹Sugarcane Experiment and Propagation Station Region 3 (Nakhon Ratchasima) Taepalai Sub-District, Khong District, Nakhon Ratchasima Province ²Cane and Sugar Promotion Center Region 3 Bangphra Sub-District, Sriracha District, Chonburi Province *Correspondence to: [email protected] ABSTRACT: : Selection on sugarcane series CSB15 in standard yield trialstage (Plant Crops and 1st ratoon) of Sugarcane Experiment and Propagation Station Region 3 (Nakhon Ratchasima) during January 2021 - February 2023 aimed to evaluate sugarcane series CSB15 of 12 elite clones; CSB15-09-05, CSB15-38-01, CSB15-09-01, CSB15-17, CSB15-20, CSB15-43, CSB15-51, CSB15-95, CSB15-155, CSB15-221, CSB15-243, and CSB15-255 compared with two standard check varieties; LK92-11 and KK3 were planted in Randomized Complete Block Design (RCBD) with four replications at two experimental fields. Sandy soil area at Phimai district, Nakhon Ratchasima province, and loamy soil area at Khu Mueang district, Buriram province. The results showed that clone CSB15-95 at 16.12 ton per rai (plant crops) and CSB15-20 at 10.94 ton per rai (1st ratoon) was the highest cane yield in sandy soil area at Phimai district, Nakhon Ratchasima province, and CSB15-09-01 at 15.38 ton per rai (plant crops) and 14.82 ton per rai (1st ratoon) was the highest cane yield in loamy soil area at Khu Mueang district, Buriram province Key Words: sugarcane breeding, conventional breeding, standard yield trial, sugarcane selection P-009 Selection on Sugarcane Series CSB15 in Standard Yield Trial Stage (Plant Crops and 1st ratoon) of Sugarcane Experiment and Propagation Station Region 3 (Nakhon Ratchasima)

105 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 an important economic crop in Thailand. In 2022/23, the production volume of sugarcane in Thailand was about 93.88 million (Office of the Cane and Sugar Board, 2023). Sugarcane is used primarily for sugar production and an efficient crop for other products, such as electricity, bioethanol and fertilizer (Unica, 2008). Despite increasing consumer demands for sugar, the cane yield and sugar yield in production systems are still low due to diseases, insect infestations and drought. Sugarcane production in Thailand is usually fluctuated due to drought. Drought causes yield reduction of 60% in more than 70% of rainfed base growing areas. (Robertson et al., 1999; Gentile et al., 2015), resulting in plant stunting and restriction of tillering, leading to vacant and low millable stalk and losses in both cane yield and sugar yield (Dinh et al., 2017). Most sugarcane varieties in Thailand have been planted for more than 15 years. Therefore, diseases and pests become virulent and can destroy old sugarcane varieties. It is necessary to develop new sugarcane varieties that are resistant to insects and diseases and tolerate drought to replace the old sugarcane varieties. The focus of The Office of Cane and Sugar Board’s (OCSB) sugarcane breeding and selection program is to obtain new high-yield varieties through breeding and selection in order to progressively, increase sugar yield in the sugarcane growing areas of Thailand. The new varieties besides high sugar yield, must adapt to the different environments and soil conditions in the production area, with genetic resistance to the main diseases, as well as adequate agronomic characteristics for their proper management. The sugarcane breeding and selection program of OCSB, genetic variability is generated through conventional breeding, establishing, mostly, bi-parental crosses using selected parents. General strategy of OCSB involves four main breeding objectives: a) sugar yield increase per unit/ area b) disease resistance, c) adaptability, and d) ratooning ability. The purpose of the study was to select sugarcane hybrid varieties at the standard yield trial stage on plant crops and 1stratoon that produced high sugarcane and sugar yields. They have the ability to adapt to drought and are resistant to diseases and insects before tested on-farm trial stage to encourage farmers to replace the old sugarcane varieties 2. MATERIALS AND METHODS Selection on sugarcane series CSB15 in standard yield trial stage (Plant Crops and 1st ratoon) of Sugarcane Experiment and Propagation Station Region 3 (Nakhon Ratchasima) during January 2021 - February 2023 aimed to evaluate sugarcane series CSB15 12 elite clone of; CSB15-09-05, CSB15-38-01, CSB15-09-01, CSB15-17, CSB15-20, CSB15-43, CSB15-51, CSB15-95, CSB15-155, CSB15-221, CSB15-243, and CSB15-255 compared with two standard check varieties; LK92-11 and KK3 were planted in Randomized Complete Block Design (RCBD) with four replications at two experimental fields. Sandy soil area at Phimai district, Nakhon Ratchasima province, and loamy soil area at Khu Mueang district, Buriram province. Subplot size 6.0 x 8.0 square meters. It has a row spacing of 1.5 meters (4 rows) and a harvest area of 3.0 x 8.0 square meters (2 middle rows). Crop management Site preparation: Prepare the soil to be suitable for sugarcane planting with a farm tractor 4 times and then raise the furrow. The furrow size is 1.50 meters wide and 30-40 centimeters deep. After planting the sugar cane, there will be 2 more cultural controls, i.e. using a spring rake to weed control before the first fertilizer application, weeding again before the second fertilizer application Sugarcane cultivation: Selecting the disease-free sugarcane setts (stalk), Cuttings sugarcane setts were to about 30 cm in length (3 buds per 1 sett) and placed in the furrow by placing two pairs of side-by-side. And cover the soil over the cultivar to a thickness of about 15-20 centimeters Fertilizer: In plant crops, first time adds fertilizer simultaneously sugarcane planting formula 16-8-8 at the rate of 50 kg per Rai, and second time adds fertilizer formula 21-7-18 when sugarcane about 4-5 months, rate 50 kg per Rai. In 1stratoon, first time adds fertilizer formula 16-8-8 at the rate of 50 kg per Rai when ratoon about 2-3 months and the last time adds fertilizer formula 21-7-18 when ratoon about 5-6 months. Data collection When the sugarcane is 12months, the data are recorded on sugar cane yield (tons perRai) and the yield component, which consists of number of millable (stalks per Rai), height (centimeters) and stalk diameter (centimeters). After recording the data on important agricultural trait, they are analyzed for sugar quality, yield sugar (t CCS. per Rai) Data analysis The variance of the data was analyzed according to the randomized complete block design (RCBD), and the mean was compared by the Least Significant Difference (LSD) method using Statistix 10 statistical analysis program.

106 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 3.1 Sugarcane Series CSB15 in standard yield trial by an average of sugarcane yield, number of millable cane and sugar yield on plant crops and 1st ratoon under rainfed condition at Phimai district, Nakhon Ratchasima province (Rainfall ; plant crops 1,527 mm., 1st ratoon 1,848 mm.) (Table 1). 3.1.1 Sugarcane Yield (t/Rai) The data show that the plant crops and the 1st ratoon are not statistically significant. 3.1.2 Number of Millable (cane/Rai) The data show a statistically significant difference for both seasons. For the plant crops, CSB15-95 (13,725 cane/Rai), CSB15-20 (13,530 cane/Rai), and CSB15-09-01 (13,129 cane/Rai) were the first three varieties to have a high number of millable. And 1st ratoon; CSB15-95 (17,817 cane/Rai) give a high number of millable, secondary CSB15-221 and CSB15-243 are an equivalent number of millable. (12,750 cane/Rai) 3.1.3 Sugar Yield (t CCS/Rai) The data show that the statistically significant difference for plant crops; KK3 (2.40 t CCS/Rai) is the highest sugar yield, CSB15-95 and CSB15-51 (1.81 and 1.71 t CCS/Rai) is the second highest sugar yield, and CSB15-09-05 (0.83 t CCS/Rai). And 1st ratoon, which is not statistically significant. 3.2 Sugarcane Series CSB15 in standard yield trial by an average of sugarcane yield, number of millable cane and sugar yield of plant crops, and 1st ratoon under rainfed condition at Khu Mueang district, Buriram province. (Rainfall ; plant crops 1,341 mm., 1st ratoon 1,633 mm.) (Table 2). 3.2.1 Sugarcane Yield (t/Rai) The data show a statistically significant difference for plant crops; the three highest yielding varieties were CSB15-09-01, CSB15-51 and CSB15-95 (15.38, 14.94 and 13.94 t/Rai respectively). And the 1st crop rotation, which was not statistically significant. 3.2.2 Number of Millable (cane/Rai) The data show a statistically significant difference for both seasons. For plant crops, the three varieties with the highest number of millable were CSB15-09-01 (15,032 cane/Rai), KK3 (12,783 cane/Rai), and CSB15-38-01 (12,774 cane/Rai). And 1st ratoon; CSB15-09-01 (14,100 cane/Rai) is the highest number of millable, LK92-11, and CSB15-38-01 (11,967 and 11,900 cane/Rai) is the second number of millable. 3.2.3 Sugar Yield (t CCS/Rai) The data show that the statistically significant difference for plant crops; CSB15-51 (1.97 t CCS/Rai) is the highest sugar yield, CSB15-255 and CSB15-95 (1.68 and 1.61 t CC /Rai) is the second highest sugar yield. And 1st ratoon that was not statistically significant difference. 3.3 Correlation analysis between 2 locations and varieties by an average of sugarcane yield, number of millable cane, and sugar yield in plant crops and 1st ratoon (Table 3). 3.3.1 Data in Table 3 showed that test planting location are not statistically significant difference for plant crops and 1st ratoon, the test planting location in Khu Muang District, Buriram Province (loamy soil); the average sugarcane yield was 11.16 t/Rai, the average number of millable cane was 15,150 cane/Rai and the average sugar yield was 1.41 t CCS/Rai which is higher than the test planting location in Phimai district, Nakhon Ratchasima province (sandy soil). Besides, the average number of millable cane is statistically significant difference for plant crops and 1st ratoon, the sugarcane varieties with a high average number of millable cane are as follows; CSB15-09-01, CSB15-221, and CSB15-95 have averages of 18,542, 16,208 and 16,071 cane/Rai, respectively and the average sugarcane yield and sugar yield are not statistically significant difference for plant crops, and 1st ratoon, The data indicate that CSB15-09-01 has the highest average sugarcane yield at 13.26 t/Rai and KK3 has the highest average sugar yield at 1.29 t CCS/Rai.

107 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 Performance of Sugarcane Series CSB15 in Standard Yield Trial by an average of cane yield, number of millable cane and sugar yield on plant crops and 1st ratoon under rainfed condition at Phimai district, Nakhon Ratchasima province ( Rainfall ; plant crops 1,527 mm., 1st ratoon 1,848 mm.) * means significant at probability level (P) < 0.05, ** means significant at probability level (P) < 0.01, and ns means not significant. The mean in the same column with the same letters is not significant. Comparison of mean by LSD at P < 0.05

108 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 2 Performance of Sugarcane Series CSB15 in Standard Yield Trial by an average cane yield, number of millable cane and sugar yield on plant crops and 1st ratoon under rainfed condition at Khu Mueang district, Buriram province. ( Rainfall ; plant crops 1,341 mm., 1st ratoon 1,633 mm.) * means significant at probability level (P) < 0.05, ** means significant at probability level (P) < 0.01, and ns means not significant. The mean in the same column with the same letters is not significant. Comparison of mean by LSD at P < 0.05

109 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 3 Performance of correlation analysis between 2 locations and varieties by an average (plant crops and 1st ratoon) of cane yield, number of millable cane and sugar yield. * means significant at probability level (P) < 0.05, ** means significant at probability level (P) < 0.01, and ns means not significant. The mean in the same column with the same letters is not significant. Comparison of mean by 4. CONCLUSIONS The result clearly showed that test planting area in Phimai district, Nakhon Ratchasima province with sandy soil characteristics, varieties with the highest sugarcane yield in plant crops and 1stratoon are CSB15-95 (16.12 t/Rai) and CSB15-20 (10.94 t/Rai), respectively. The varieties with high sugar yield are nearly standard check varieties in plant crops and 1st ratoon CSB15-95 (1.81 t CCS/Rai) and CSB15-20 (1.51 t CCS/Rai), respectively.

110 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 Khu Muang district, Buriram province with sandy soil characteristics, variety with the highestsugarcane yield in plant crops and 1st ratoon are CSB15-09-01 (15.38 and 14.82 t/Rai), respectively. The varieties with high sugar yield are nearly the standard check varieties in plant crops and 1st ratoon CSB15-51 (1.97 t CCS/Rai) and CSB15-09-01 (1.64 t CCS/Rai), respectively. Correlation analysis between two locations and varieties by an average of sugarcane yield, number of millable cane, and sugar yield in plant crops and 1st ratoon found that the test planting area in Khu Muang district, Buriram province with loamy soil characteristics. There is average sugarcane yield, number of millable cane, and sugar yield is higher than the test planting area in Phimai district, Nakhon Ratchasima province with sandy soil characteristics. 5. RECOMMENDATION The sugarcane yield, quality and yield components of sugarcane production. It also depends on the distribution of rainfall per year. When rainfall is insufficient, a management plan must be made to prepare the soil appropriately, such as plowing the soil to collect rainwater and increasing the amount of organic matter in the soil. Soils with a high percentage of organic matter (OM) and aggregates can absorb and store water during rainfall and release it to the plants during the dry season. In this way, sugarcane can grow and produce even under drought conditions. 6. REFERENCES [1] Dinh HT, Watanable K, Takaragawa H, Kawamitsu Y. (2017). Effects of drought stress at early growth stage on response of sugarcane to different nitrogen application. Sugar 482 Tech doi: 10.1007/s12355-017-0566-y [2] Gentile A, Dias LI, Mattos RS, Ferreira TH, Menossi M. (2015). MicroRNAs and 494 drought responses in sugarcane. Frontiers in Plant Science 6, 1-13, doi: 10.3389/fpls.2 495 015.00058 [3] Office of the Cane and Sugar Board (2022). Final report of sugar and cane production in Thailand 2021/2022. http://www.sugarzone.in.th/ Accessed 19 April 2023 [4] Robertson MJ, Inmam-Bamber NG, Muchow RC, Wood AW. (1999). Physiology and productivity of sugarcane with early and mid-season water deficit. Field Crops Research 64, 211–227, doi: 10.1016/ S0378-4290(99)00042-8 [5] Unica. (2008). Sugarcane industry in Brazil. http://sugarcane.org/resource-library/books/UNIC 571 As%20 Institutional%20Folder.pdf. Accessed 15 June 2023.

111 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 Raweewan Chuekittisak1 *, Sontaya Khamtib2 , Ratchada Pruscharoenwanich3 , Weerakorn Saengsai¹, Wassana Wandee¹ and Nattapat Khumla¹ ¹Field and Renewable Energy Crops Research Institute, Bangkok. ²The Agricultural Production Factors Research and Development Division, Bangkok. ³Nakhon Ratchasima Agricultural Research and Development Center, Nakhon Ratchasima. *Correspondence to: Field and Renewable Energy Crops Research Institute, Department of Agriculture, [email protected] ABSTRACT: Energy is an important sector in driving the country’s economy and has fluctuating prices. Thailand imports energy approximately 60 percent of the usual consumption so it is necessary to be more self-supported by seeking alternative sources of energy which is environmental-friendly. Sugarcane is an important economic crop for producing sugar and can be processed into various forms of energy. Ethanol is a form of energy that can be produced from both sugar cane and bagasse and the suitable varieties for energy has not been discovered. A study was conducted to find out the potential in ethanol production of the bio-energy sugarcane clones in the standard yield trial experiments in 2022 at Khon Kaen Field Crops Research Center, Nakhon Ratchasima Agricultural Research and Development Center and the Agricultural Production Factors Research and Development Division. The objective was to find out the sugarcane clones providing higher ethanol production by using the yeast Saccharomyces cerevisiae SK19 in batch fermentation. In planting cane, it was found that varieties K88-92 and Khon Kaen 3 (KK3) provided the highest ethanol production from both sugarcane juice and bagasse theoretically, however, there werebthe promising clones producing as much ethanol as the two varieties, KK13-203, KK07-250 and UTe05-110. On the contrary, the experimental ethanol yield from the juice of clones KK07-250, KK11-158, KK12-050 and KK12R-076 showed higher than that of the checked varieties. The theoretical ethanol yield from bagasse was found as high as 10,481 liters/ha different from the experimental ethanol yield, which was 638 liters/ rai or only 2.9 percent. KK3 and K88-92 varieties provided the highest both theoretical and experimental ethanol production from bagasse. The promising clones KK07-250, KK13-171 and KK13-330 showed no different ethanol production from KK3. In addition, the clones KK13-203, UTe05-110, UTe05-112 and KK07-250 provided as high ethanol production from bagasse as K88-92 varieties. Keyword: ethanol yield, batch fermentation, bagasse, juice P-011 Ethanol Production Potential for Promising Sugarcane Clone

112 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 reduction in fossil-based petroleum production and the war between Russia and Ukraine caused the instability of the petroleum oil supply. Consequently, there have been efforts to seek more sustainable alternative energy sources and be environmentally friendly by reducing greenhouse gas emissionsfrom fuel combustion. Ethanol is a high-octane renewable energy source, clean and completely combustible affecting in reducing air pollution. Ethanol can be processed from plant-based materials and continuously renewed. Growing crops are capable to absorb the amount of carbon dioxide in the atmosphere. It is used to mix with gasoline, called gasohol, mix with diesel fuel, called diesohol. and pure ethanol can be used as fuel directly. It is mainly produced from sugarcane and cassava in Thailand. The ethanol from sugarcane is produced using molasses and cane juice. These consisted of 11 factories that produced ethanol from molasses and only one factory produced cane juice, with a total production capacity of (12 factories) about 500,000 liters per day. The raw materials of 0.35 million tons of molasses and 100 million tons of sugarcane were needed annually. It has been realized that sugarcane is an essential economic crop in producing alternative energy. However there has not been any sugarcane cultivar specifically for renewable energy production even though the Department of Agriculture has been conducting research and development on sugarcane varieties continually. Therefore, the ethanol production potential of the promising sugarcane clones was studied in Standard Trial at Khon Kaen Field Crops Research Center (KKFCRC), Nakhon Ratchasima Agricultural Research and Development Center (NRARDC) in 2021-2023 and the Agricultural Production Factors Development Research Division (APFDRD) is concerned with the analysis of ethanol production potential. The objective was to find out promising sugarcane clones with high ethanol yield potential. 2. MATERIALS AND METHODS 2.1 Standard Trials were implemented in 2021-2022 at KKFCRC and NRARDC, 1 plot each. There were 3 replications in RCB experiment design, consisting of promising 19 varieties/clones (TPJ04-768, UTe05-102, UTe05-110, UTe05-112, KK07-250, KK07-599, KK11-658, KK12-050, KK12R-076, KK13-114, KK13-171, KK13-203, KK13-330, KK13-470, KK13-483 with 4 checks of U-Thong 2 (UT2), Khon Kaen 3 (KK3), K88-92 and LK92-11) in each experimental site. Yield and yield components were collected at 10 months. 2.2 Study on the ethanol yield potential of sugarcane promising clones was conducted at APFDRD laboratory in 2021-2022. The yeast seedling culture of Saccharomyces cerevisiae SK-19 was prepared by incubating yeast malt medium at 35 °C for 48 hours. Then sugarcane juice was squeezed out and separated bagasse. The juice was analyzed for total sugar content. The bagasse was chopped into small size and analyzed for the composition, namely cellulose, hemicellulose and lignin then stored at -20 °C for preservation and maintaining specific constituents throughout the experiment. The sugarcane juice, bagasse and chopped bagasse with sugarcane juice were processed into ethanol by batch fermentation with Saccharomyces cerevisiae SK-19 yeast cultures, incubated at 35-37 °C, and shaken on an incubator at 150 rpm. Samples were collected every 4 hours and analyzed the ethanol content with High-Performance Liquid Chromatography (HPLC) using Aminex HPX-87H column. Total sugar content was analyzed with the phenol-sulfuric acid method, total solids (TS) and volatile solids (VS) were analyzed according to APHA standard methods. (APHA, 1997) Ethanol yield (Yp/s) is calculated from the amount of ethanol produced in grams per gram of sugar used, while the ethanol productivity, Qp, was measured in grams per liter per hour (Equation 1) and yield efficiency of ethanol production, Ey, was calculated in Equation 2 as follows: Qp = P/t ………………………………….. (1) Ey = (Yp/s × 100) / 0.538 ……………….. (2) where P is the concentration of ethanol produced, in grams per liter. t is the maximum ethanol concentration time in hours. 0.538 is the theoretical maximum yield of ethanol production from sucrose 3. RESULTS AND DISCUSSION 3.1 Sugarcane yield trial: Standard Trial at KKFCRC Sugarcane was harvested at the age of 10 months on November 8, 2022. K88-92 varieties provided the highest yield of 129.3 tons/ha, however, not statistically different from cultivars/clones KK07-250, KK07-599, KK12-050, KK13-203, KK13-330, LK92-11, UT2 and KK3. Sugarcane clone KK13-330 showed the highest number of stalks and stools harvested but less stalk length and diameter leading to lower cane yield. The stalk weight and length were not statistically different. At NRARDC, it was found that KK13-203 gave the highest cane yield of 220.6 tons/ha, not statistically different

113 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 from KK3 and K88-92. Moreover, KK13-203 had the highest stalk weight of 2.79 kg/stalk. Clone KK13-330 showed the highest number ofstalks, 148,831 stalks/ha, but gave not much stalk length and weightso the cane yield waslower. Clone UTe05-110 had the longest stalk diameter of 3.19 cm, not statistically different from KK07-250, KK07-599, KK13-203 and KK13-483. The stalk weight and length were not statistically different. It showed that the data of KKFCRC and NRARDC could be analyzed the covariance for 3 parameters, cane yield, number of stalks and stalk diameter, whereas stalk weight and length and number of internodes per stalk could not. Clone KK13-203 provided the highest average cane yield of 158.7 tons/ha, higher than that of K88-92 and KK3. The clones KK13-330, UTe05-112 and KK12R-076 provided a higher number of stalks harvested (106,875 75,488 and 68,959 stalks/ha, respectively) than KK3 (68,956 stalks/ha). There were 2 promising clones giving higher stalk numbers than 4 standard varieties, namely KK07-599 and KK13-203, both giving 2.61 kg/stalk. The sugarcane clone KK13-114 had longer stalk length than the 4 standard varieties. There were 5 clones providing higher stalk diameters than the standard varieties, namely UTe10-110, KK07-250, KK07-599, KK13-203 and KK13-483. (Table 1) 3.2 Ethanol yield potential of sugarcane promising clones. In the ethanol production process from juice, it was shown that the ethanol concentration of the sugarcane clones tested was ranged from 13.56-78.11 g-EtOH/L (the 4 checked varieties ranged from 12.08-72.54 g-EtOH/L), ethanol productivity (Qp) was in the range of 0.28-1.63 g-EtOH/L h (the 4 checked varieties ranged 0.25-1.51 g-EtOH/L h), ethanol yield (Yp/s) was in the range of 0.09-0.49 g-EtOH/g-Sugar consumed (the 4 checked varieties ranged 0.09-0.38 g-EtOH/g-Sugar consumed), ethanol yield efficiency (Ey) was in the range of 17.1-90.3% of the theoretical ethanol yield (the 4 checked varieties ranged 15.8-70.8%). In the ethanol production from bagasse, it was found that ethanol concentration lined in the range of 0.16-0.63 g-EtOH/L (the 4 checked varieties ranged 0.32-0.63 g-EtOH/L), ethanol productivity (Qp) ranged from 0.03-0.13 g-EtOH/L d (the 4 checked varieties ranged 0.06-1.13 g-EtOH/L d), ethanol yield (Yp/s) ranged from 0.005-0.022 g-EtOH/g-dry bagasse (the 4 checked varieties ranged 0.011-0.024 g-EtOH/g-dry bagasse) and ethanol yield efficiency (Ey) ranged from 1.34.7-5.49% (the 4 checked varieties ranged 2.67-5.96 %) (Table 2). In the case of the potential of sugarcane juice and bagasse in ethanol production of 19 clones/varieties, it showed that sugarcane variety K88-92 provided the highest ethanol yield from juice and bagasse theoretically of 5,244 and 10,481 liters/ha (summed up to 15,725 liters/ha) at KKFCRC but its ethanol yield from juice and bagasse experimentally were not so high, 769 and 638 liters/ha (summed up to 1,407 liters/ha), respectively. It also showed that UT2 variety gave the highest ethanol yield efficiency (Ey) of 30.2%, the highest ethanol yield from juice experimentally and a total ethanol yield of 3,275 and 3,625 liters/ha, respectively (Table 3). At NRARDC, KK3 variety gave the highest ethanol yield from sugarcane juice and bagasse of 8,775 and 18,663 liters/ha theoretically (summed up to 27,438 liters/ha) but the clone KK13-203 provided the highest ethanol yield from juice experimentally of 6,969 liters/ha, moreover, K88-92 gave highest ethanol yield from bagasse experimentally of 838 liters/ha. The promising clones showing higher total ethanol yield efficiency (Ey) were KK11-158, KK13-203, UTe0-110 and UTe05-102, giving 32.8 32.6 26.9 and 26.1%, respectively (Table 3). As averaged of the 2 sites, it was found that theoretical ethanol yield from juice was highest in the standard varieties K88-92 and KK3 but the experimental one was higher in clones KK13-203, KK07-250, KK11-158, KK12-050 and KK12R-076 (5,044 3,719 3,663 3,469 and 3,463 liters/ha, respectively). KK3 gave the highest theoretical ethanol yield from bagasse (13,144 liters/ha), higher than that of K88-92 (12,150 liters/ha), but gave lower experimental ethanol yield from bagasse, 369 liters/ha for KK3 and 738 liters/ha for K88-92. The promising clones which gave higher theoretical ethanol yield from bagasse than K88-92 were KK13-330, KK07-250, KK13-171, KK07-599 and the ones giving higher experimental ethanol yield from bagasse than KK3 consisted of KK13-203 and Ute05-110 (Table 3). The promising clones providing ethanol yields close to that of K88-92 were KK13-203, UTe05- 110, UTe05-112 and KK07-250. Most of them had high ethanol yield efficiency. The theoretical and experimental ethanol yield from bagasse will be very different, the theoretical will give an average ethanol yield of 10,481 liters/ha, but the experiment yields 300 liters/ha representing 2.9%. 4. CONCLUSION The promising sugarcane clones which showed high potential ethanol production from juice and bagasse (at 10 months old harvest) were composed of UTe05-110, UTe05-112, KK07-250, KK11-158, KK12-050, KK12R-076 and KK13-203. These would be used as genetic resources for energy cane varietal development and an alternative energy crop for farmer community enterprises and power plants in the future. 5. ACKNOWLEDGEMENT The research team would like to send many thanks to the Director of Field and Renewable Energy Crops Research Institute, the Director of Khon Kaen Field Crops Research Center, the Director of Nakhon Ratchasima

114 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 Agricultural Research and Development Center, the Director of The Agricultural Production Factors Research and Development Division and their concerned government employees and agricultural experiment workers. Finally, we thank the National Research and Innovation Information System (NRIIS) and The Japan International Research Center for Agricultural Sciences (JIRCAS) for supporting the research budget. 6. REFERENCES APHA. 1995. Standard methods for the examination of water and wastewater. 19th Edition. American Public Health Association, Washington, DC.

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118 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 Supot Kasem1 *, Atithan Paythai1 , Trairong Yodgeaw¹, Tiyakorn Chatnaparat¹ 1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900 THAILAND *Correspondence to: Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900 THAILAND ABSTRACT: Red rot disease caused by Fusarium moniliforme is an important diseases ofsugarcane causing considerable economic loss in Thailand. This disease was negatively affects cane yield and sugar quality. Chemicals and cultural practices were limitation for disease control. Biological control using antagonistic bacteria can be used as combination and alternative to chemicals. The efficacy of different antagonistic bacterial strains on growth inhibition of F. moniliforme under laboratory conditions was tested. Bacillus amyloliquefaciens S20A1 and KPS46 showed highest effective to inhibit growth of target pathogen with inhibitory activities at 34.22 and 31.11% followed by B. velenzensis KN. Under greenhouses conditions, effect of soaking stem cutting (cv. Khon Kaen 3) before growing, and 3-time soil drench at 7 days intervals with different bacterial cell suspensions at 7 weeks-olds sugarcane seedling was evaluated. The results revealed that all of bacterial antagonist strain could control red rot disease with severity reduction ranging from at 91.33-92.52%. These bacterial antagonists also showed the potential on induction of defense enzyme accumulation in treated plant with related to disease reduction. B. amyloliquefaciens KPS46 showed the most effective on induced plant to accumulate Peroxidase (POX) and Phenylalanine ammonia-lyase (PAL) when B. amyloliquefaciens S20A1 was highest effective on induced plant to accumulate β-1,3 glucanase. These two defense related enzymes were highest express at 3 days after challenge inoculation with the causal pathogen. Keyword: Sugarcane diseases, Fusarium moniliforme, Biocontrol mechanism, induced plant resistance P-012 Control Efficacy of Antagonistic Bacteria Bacillus amyloliquefaciens Strain S20A1 and KPS46 Against Red Rot Disease in Sugarcane

119 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 Jutatape Watcharachaiyakup1,2*, Supananya Chansri1,2, Thanyaluk Thaitae1,2, Pimpilai Saengmanee1,2, and Parichart Burns³ ¹Center for Agricultural Biotechnology, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand ²Center of Excellence on Agricultural Biotechnology: (AG-BIO/MHESI), Bangkok 10900, Thailand ³National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Correspondence to: Center for Agricultural Biotechnology, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand. [email protected] ABSTRACT: Sugarcane white leaf phytoplasma (SCWL) has been a significant disease in Thailand since its first found in 1954. The genetic diversity of SCWL in Thailand has been previously reported using 16S rDNA, secA, and leuS genes. This current study aimed to investigate the diversity of SCWL based on the groES gene region, which has not been previously explored. A total of 205 SCWL samples were collected from ten provinces in Thailand, including Kamphaeng Phet and Utai Thanee in the North, Kanchanaburi, Phetchaburi, and Prachuap Khiri Khan in the Central area, and Udon Thani, Kalasin, Mukdahan, Roi Et, and Surin in the Northeastern region. Polymerase chain reaction (PCR) was used to amplify the groES gene region, and Sanger sequencing was used to directly determine the nucleotide sequences. The resulting sequences were quantified, aligned, and clustered by maximum likelihood, Neighbor-Joining and Minimum-evolution method to assess the genetic diversity. The analysis revealed only four nucleotide differences out of a total of 210 nucleotides. The results of the clustering analysis demonstrate that the samples collected from the SCWL in Thailand can be segregated into two distinct subgroups, with a high correlation observed across all three calculation methods utilized. Keyword: genotyping, sugarcane, disease P-016 groES gene diversity among sugarcane white leaf phytoplasma in Thailand

120 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 Parichart Burns¹, Pimpilai Saengmanee2,3, Jutatape Watcharachaiyakup2,3, Udomsak Lertsuchatavanich⁴ and Sonthichai Chanpreme⁵ 1 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand ²Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom 73140, Thailand ³Center of Excellence on Agricultural Biotechnology: (AG-BIO/MHESI), Bangkok 10900, Thailand ⁴Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkhen 10900, Thailand ⁵Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand *Correspondence to: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand E-mail [email protected] ABSTRACT: Sugarcane white leaf (SCWL) phytoplasma (classified as Candidatus Phytoplasma sacchari) is a major pathogen causing major lossesin sugarcane production. Symptoms of this disease range from mild to severe with characteristics of white leaf, stunting and grassy shoot. There is little known of mechanisms related to disease virulence in plant host. ATP dependent zinc metalloprotease (FtsH) is a protease assisting in protein insertion and stabilization in bacterial membrane. Studies in bacteria including Pseudomonas aeruginosa and Staphylococcus aureus indicated that FtsH played important roles in pathogenicity and their amino acid variations altered their pathogenicity. In this study, Ca. Phytoplasma sacchari FtsH Thai isolate was cloned, sequenced and their protein structure investigated. The FtsH was 625 amino acids in length. It has conserved motifs characteristics of bacteria FtsH including two transmembrane helices, Walker A motif, Walker B motif, pore residues, second region of homology (SRH) and zincin HEXXH motif and leucine-rich motif. Analysis of FtsH amino acid sequence variation showed that four amino acid changes possibly altered FtsH function. Two of them were located extracellular and between two transmembrane domains. One alteration occurred near zincin HEXXH motif which could alter protease activity. Keyword: protease, transmembrane protein, degradation P-019 Amino acid changes in ATP dependent zinc metalloprotease, a protein stabilizer, in SCWL phytoplasma Thai isolate in 3D structure and the potential implication

121 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 ATP dependent zinc metalloprotease (FtsH) is an enzyme found in eubacteria, mitochondria and chloroplast. FtsH was approx. 650 amino acids in length. FtsH proteins assembled into hexamers with conserved arginine in SRH believed to be critical for FtsH oligomerization [1]. Three genera in class mollicutes including mycoplasmas, spiroplasmas and acholeplasmas had a single copy of FtsH [2-4] however many phytolasmas consisted of multiple copies of FtsH. Candidatus Phytoplasma mali had four copies of FtsHs [5]. Nine copies of FtsHs were identified in Ca. P. ziziphini [6]. The functions of bacterial FtsH included degradation of membrane proteins, regulation of heat shock response, lipopolysaccharide biosynthesis, superoxide stress response and virulence [7]. The role of FtsH in virulence was investigated in pathogenic bacteria including Pseudomonas spp. and Salmonella spp. [8-9]. The function of FtsHs in Candidatus Phytoplasma in virulence was also described. Seemüller et al [10] reported that FtsH of Ca. Phytoplasma malirelated to disease virulence in apple trees.The phylogenetic diversity and membrane topology ofCa. Phytoplasma mali FtsH and the relationship to strain virulence was also further demonstrated [5]. Sugarcane white leaf disease is caused by Ca. Phytoplasma sacchari. The symptoms of disease varied from mild to severe. The latter causes major losses in sugarcane production. There were little studies into the mechanisms and proteins that related to disease virulence. In this study, protein structure of FtsH from Ca. Phytoplasma sacchari was analyzed. 2. MATERIALS AND METHODS DNA sequence encoded a FtsH protein of Ca. Phytoplasma was amplified from sugarcane cv. Khon Khen 3 sample with white leaf symptom from Chonburi province. The DNA fragments were cloned and sequenced. FtsH amino acid sequence ofCa. Phytoplasma sacchariThai isolate was compared to Phytoplasma FtsH reference sequences from Genbank database using CLUSTALW. The topology of transmembrane proteins was predicted using CCTOP (https://cctop.ttk.hu/) [11]. The localization of a putative alpha-helical transmembrane regions and the orientation were predicted by 10 different methods using available structural and experimental information in the Topology Data Bank of Transmembrane Proteins (TOPDB) database. Prediction of amino acid changes that affect protein function was performed using SIFT (https://sift.bii.a-star.edu.sg/www/SIFT_seq_submit2.html) [12]. The program processed query sequences in 4 stages: gathering homologous sequences, fold library scanning, loop modelling and side chain placement. Amino acid sequence and substitution of interest were submitted into Sort intolerant to tolerant (SIFT) SIFT. They were compared with protein sequences in UniProt-SwissProt and TrEMBL 2010_09. 3. RESULTS AND DISCUSSION FtsH of Ca. Phytoplasma sacchari Thai isolate was 625 amino acids in length. The molecule weight was 70.7 kDa and pI = 9.68. The protein possessed motifs, which are characteristics of bacterial FtsH. The N-terminal part consisted of 2 transmembrane helices that anchored into lipid bilayer membranes. The transmembrane helices connected to AAA moiety through a linker with 15-20 amino acids in length. AAA moiety (also called ATPase domain) contained Walker A motif, Walker B motif, pore residues and second region of homology (SRH) fingerprint. The protease domain at C-terminus contained zincin HEXXH motif and leucine-rich motif (Figure 1) [13]. The location scores of inner membrane and cytoplasmic membrane were 1.486 and 5.402, respectively. CLUSTALW multiple sequence alignment showed that a Thai Ca. Phytoplasma sacchari FtsH had high homology of 98.8% to Ca. Phytoplasma sacchari accession number WP153369138 with 7 amino acid differences followed by 79.0% homology with Ca. Phytoplasma oryzae accession number CP116038 (Figure 2). A linker with 20 amino acid residue in length between the second transmembrane helix and cytosolic domain was found to be conserved between Ca. Phytoplasma sacchari and Ca. Phytoplasma oryzae (Figure 3). The linker was essential for proteolytic activity of FtsH [1]. Due to similarity in symptoms in rice yellow dwarf disease [14] and sugarcane white leaf disease, FtsH might play role in symptomatic development in both phytoplasma. Using CCTOP, the location of two transmembrane regions was identified. HMMTOP and Phobius provided identical results while Memstat and TMHMM only predicted one transmembrane region. Octopus, on the other hand, identified 4 transmembrane regions (Figure 4). Amino acid changes in FtsH (from Thai isolate) that could possibly affect protein function were predicted with SIFT by comparing with two Ca. Phytoplasma sacchari sequences (WP153369138, from India and CP115156, from China). Sugarcane samples from Thailand and China showed characteristic of white leaf symptom while the sample from India displayed grassy shoot symptom. Four amino acid changes were found to affect the protein function. These included threonine to lysine at position 57, threonine to asparagine at position 89, valine to alanine at position 461 and serine to glycine at position 538. The first two changes caused an alteration of charge group and located in the region between two transmembrane helices, possibly extracellularly. The position 461 was adjacent to protease motif.

122 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 latter amino acid change in FtsH could use to distinguish between Ca. Phytoplasma sacchari isolates causing sugarcane white leaf symptom (obtained from Thailand and China) and the isolate causing grassy shoot symptom in sugarcane (from India) [15]. Figure 1 Schematic diagram of Ca. Phytoplasma sacchari FtsH. It included two transmembrane helices, ATPase domain and protease domain. Figure 2 Phylogenetic tree from ClustalW multiple sequence alignment of phytoplasma FtsHs. The FtsH reference sequence included Ca. Phytoplasma sacchari (accession number WP153369138), Ca. Phytoplasma oryzae (accession number CP116038), CP015149 (Ca. Phytoplasma asteris-maize bush stunt phytoplasma), CP040925 (Echinacea purpurea witches’ broom phytoplasma), CP000061 (Aster yellows witches’ broom phytoplasma), NC011047 (Ca. Phytoplasma mali). Figure 3 Prediction of transmembrane domains of FtsH Thai isolate using CCTOP. Two transmembrane domains at position 11-30 and 132-152 (yellow) were predicted. The identical regions were also predicted by HMMTOP, Octopus and Phobius. Octopus also predicted two additional regions. Memstat and TMHMM only predicted one transmembrane region (3a). The locations of two transmembrane helices were shown in (3b) and (3c). Figure 4 ClustalW alignment of FtSHs showing a 20 amino acid linker (grey highlight) conserved in Ca. phytoplasma sacchari Thai isolate and Ca. Phytoplasma oryzae (accession number CP116038). FtsH reference sequences also included CP015149 (Ca. Phytoplasma asteris-maize bush stunt phytoplasma), CP040925 (Echinacea purpurea witches’ broom phytoplasma), CP000061 (Aster yellows witches’ broom phytoplasma), NC011047 (Ca. Phytoplasma mali).

123 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. CONCLUSIONS ATP dependent zinc metalloprotease (FtsH) from Ca. Phytoplasma saccahri protein structure was investigated. It consists of two transmembrane helices and conserved motifs of ATPase domain and protease domain. The sequence showed high homology to Ca. phytoplasma oryzae. Four amino acid variation among three Ca. Phytolasma sacchari FtsHs could affect protein function. Two variations located between transmembrane helices while a variation located near ZIN motif in protease domain. 5. ACKNOWLEDGEMENT The authors would like to thank National Research Council of Thailand and The Integrated Research Office for sugarcane and sugar projects for their financial support and valuable suggestion. 6. REFERENCES [1] Carvalho V, Prabudiansyah I, Kovacik L, Chami M, Kieffer R, Van Der Valk R, De Lange N, Engel A. and Aubin-Tam ME. (2021). The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry. Journal of Biological Chemistry, p.296, DOI: 10.1074/jbc. RA120.014739 [2] Staats CC, Boldo J, Broetto L, Vainstein M. and Schrank A. (2007). Comparative genome analysis of proteases, oligopeptide uptake and secretion systems in Mycoplasma spp. Genetics and Molecular Biology, 30, pp. 225-229, DOI: 10.1590/S1415-47572007000200009 [3] Kube M, Schneider B, Kuhl H, Dandekar T, Heitmann K, Migdoll AM, Reinhardt R. and Seemüller E. (2008). The linear chromosome of the plant-pathogenic mycoplasma 'Candidatus Phytoplasma mali'. Bmc Genomics, 9(1), pp.1-14, DOI: 10.1186/1471-2164-9-306 [4] Kube M, Siewert C, Migdoll AM, Duduk B, Holz S, Rabus R, Seemüller E, Mitrovic J, Müller I, Büttner C. and Reinhardt R. (2014). Analysis of the complete genomes of Acholeplasma brassicae, A. palmae and A. laidlawii and their comparison to the obligate parasites from ‘Candidatus Phytoplasma'. Microbial Physiology, 24(1), pp.19-36, DOI: 10.1159/000354322 [5] Seemüller E, Sule S, Kube M, Jelkmann W. and Schneider B. (2013). The AAA+ ATPases and HflB/FtsH proteases of ‘Candidatus Phytoplasma mali’: phylogenetic diversity, membrane topology, and relationship to strain virulence. Molecular Plant-Microbe Interactions, 26(3), pp.367-376, DOI: 10.1094/MPMI-09-12-0221-R [6] Xue C, Zhang Y, Li H, Liu Z, Gao W, Liu M, Wang H, Liu P. and Zhao J. (2023). The genome of Candidatus phytoplasma ziziphi provides insights into their biological characteristics. BMC Plant Biology, 23(1), pp.1-13, DOI: 10.1186/s12870-023-04243-6 [7] Langklotz S, Baumann U. and Narberhaus F. (2012). Structure and function of the bacterial AAA protease FtsH. Biochimica Et Biophysica Acta (BBA)-Molecular Cell Research, 1823(1), pp.40-48, DOI: 10.1016/j. bbamcr.2011.08.015 [8] Kamal, S.M., Rybtke, M.L., Nimtz, M., Sperlein, S., Giske, C., Trček, J., Deschamps, J., Briandet, R., Dini, L., Jänsch, L. and Tolker-Nielsen, T., 2019. Two FtsH proteases contribute to fitness and adaptation of Pseudomonas aeruginosa clone C strains. Frontiers in Microbiology, 10, p.1372, DOI: 10.3389/ fmicb.2019.01372 [9] Choi E, Kwon K. and Lee EJ. (2015). A single amino acid of a Salmonella virulence protein contributes to pathogenicity by protecting from the FtsH-mediated proteolysis. FEBS letters, 589(12), pp.1346-1351, DOI: 10.1016/j.febslet.2015.04.014 [10] Seemüller E, Kampmann M, Kiss E. and Schneider B. (2011). HflB gene-based phytopathogenic classification of ‘Candidatus Phytoplasma mali’strains and evidence that strain composition determines virulence in multiply infected apple trees. Molecular plant-microbe interactions, 24(10), pp.1258-1266, DOI: 10.1094/ MPMI-05-11-0126 [11] Dobson L, Reményi I. and Tusnády GE. (2015). CCTOP: a Consensus Constrained TOPology prediction web server. Nucleic acids research, 43(W1), pp. W408-W412, DOI: 10.1093/nar/gkv451 [12] Ng PC. and Henikoff S. (2003). SIFT: Predicting amino acid changes that affect protein function. Nucleic acids research, 31(13), pp.3812-3814, DOI: 10.1093/nar/gkg509 [13] Vostrukhina M, Popov A, Brunstein E, Lanz, MA, Baumgartner R, Bieniossek C, Schacherl M. and Baumann U. (2015). The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer. Acta Crystallographica Section D: Biological Crystallography, 71(6), pp. 1307-1318, DOI: 10.1107/ S1399004715005945 [14] Kumar M, Wu Y, Gu P, Hoat TX, Valarmathi P. and Rao GP. (2023). Updates on phytoplasma diseases associated with cereals in Asia. In Phytoplasma Diseases of Major Crops, Trees, and Weeds, pp. 1-18. Academic Press, DOI: 10.1016/B978-0-323-91897-8.00018-6 [15] Kirdat K, Tiwarekar B, Thorat V, Narawade N, Dhotre D, Sathe S, Shouche Y. and Yadav A. (2020). Draft genome sequences of two phytoplasma strains associated with sugarcane grassy shoot (SCGS) and bermuda grass white leaf (BGWL) diseases. Molecular plant-microbe interactions, 33(5), pp.715-717, DOI: 10.1094/ MPMI-01-20-0005-A

124 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 Parichart Burns1 *, Jutatape Watcharachaiyakup2,3, Udomsak Lertsuchatavanich⁴, Praderm Wanichananan¹, Sutticha Na-Ranong Thammasittirong⁵, Patchima Sithisarn⁶, Pimpilai Saengmanee2,3, Supattana Chanta¹ and Sonthichai Chanpreme⁶ ¹National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand ²Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom 73140, Thailand ³Center of Excellence on Agricultural Biotechnology: (AG-BIO/MHESI), Bangkok 10900, Thailand ⁴Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkhen 10900, Thailand ⁵Dept. of Veterinary Public Health, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand ⁵Microbial Biotechnology Unit, Faculty of Veterinary medicine, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand ⁶Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand *Correspondence to: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand. E-mail [email protected] ABSTRACT: Sugarcane white leaf phytoplasma is an important pathogen causing majorlossesin sugarcane production. The infected sugarcane without symptom can spread the pathogen into new plantation. Hence, the control and management of this disease is difficult. Therefore, it is important to investigate the impact of this pathogen to sugarcane host in order to effectively control the pathogen and determine changes in quality and quantity of the product. The BIOTEC-KU cooperation team with members with expertise in plant pathology, plant molecular biology, chemical identification and agronomy was formed and funded by Cane and sugar research coordination under National Research Council of Thailand (NRCT). The aims of our research are to conduct research and development on SCWL phytoplasma diversity, rapid detection and plant-pathogen interaction with the goals of 1) effectively and rapidly monitoring and managing SCWL phytoplasma and 2) enhance effectiveness ofsugarcane production.The research has made important finding and progresses including SCWL phytoplasma diversity in Thailand and impacts of SCWL phytoplasma to biochemical compositions of sugarcane. Keyword: sugarcane phytoplasma plant-pathogen interaction genetic diversity P-020 Progress research on sugarcane white leaf phytoplasma by BIOTEC-KU team (2020-2023)

125 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 Pimpilai Saengmanee1,2*, Parichart Burns³, Jutatape Watcharachaiyakup1,2, Udomsak Lertsuchatavanich⁴, and Sonthichai Chanpreme5 1 Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom 73140, Thailand ²Center of Excellence on Agricultural Biotechnology: (AG-BIO/MHESI), Bangkok 10900, Thailand ³National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand ⁴Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkhen 10900, Thailand ⁵Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand *Correspondence to: Pimpilai Saengmanee Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom 73140, Thailand. E-mail [email protected] ABSTRACT: PCR based detection is one of the most effective techniques for SCWL phytoplasma detection. Nucleic acids were extracted from SCWL infected sugarcane using EPPO Diagnostic Standard of phytoplasmas. The specificity and sensitivity of six phytoplasma primers was determined. 16-23SrRNA and SecA showed better performance than others. Though 16s-23 rDNA primers were more sensitive, nested-PCR wasrequired forspecific amplification of SCWLphytoplasma. SecAprimers, on the other hand, were less sensitive for SCWL amplification. Sequence analysis showed deletion and sequence variation at forward primer region of SecA primer. Therefore, SecA-SCWL primers was developed and found to be sensitive and SCWL phytoplasma specific. Forty-one asymptomatic and 19 symptomatic Khon Kaen 3 (KK3) sugarcane samples from Bueng Samakkhi District, Kamphaeng Phet province Thailand were used as template DNA for amplification. 16-23SrRNA and SecA-SCWL primers could detect SCWL phytoplasma in both asymptomatic and symptomatic sugarcane samples. The sensitivity range was between 0.01 ng -1 microgram. Keyword: 16sRNA, SCWL phytoplasma, sugarcane P-022 Specificity and sensitivity of sugarcane white leaf (SCWL) phytoplasma molecular detection

126 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 Piyanan Thanomchat1 *, Yupadee Paopun1 and Nustsaba Kronburee1 ¹Scientific Equipment and Research Division, Kasetsart University research and Development Institute, Bankok, 10900, Thailand *Correspondence to: Scientific Equipment and Research Division, Kasetsart University research and Development Institute, 50 Ngamwongwan Road, Ladyao, Chatujak, Bangkok 10900, Thailand. [email protected] ABSTRACT: Sugarcane (Saccharum officinarum L.) belongs to the family Graminae or Poaceae. It is an economic crop of Thailand. Which is used as a raw material in the cane and sugar industry. The aim of this research was to examine characteristic, distribution and density of stomata, silica cell and cork cell on adaxial and abaxial leaf surfaces of five sugarcane cultivars in Thailand in order to evaluate drought tolerant of sugarcane cultivars. In addition, silica content in elite cultivars is important to silical industry. Sugarcane cultivars including KK3, KPS01-12, LK92-11, UT84-12 and SU50 were selected for this study. The adaxial and abaxial leaf surfaces were observed using a light microscopy (LM) and field-emission scanning electron microscopy (FE-SEM). The results showed significant different of stomatal size and stomatal density of both surfaces among the 5 cultivars. The SU50 cultivar has the highest stomatal length on adaxial and abaxial leaf surfaces (38.63µm±1.66 and 40.84 µm±1.91 respectively). The stomatal type is exclusively paracytic with two subsidiary cells. The UT84-12 cultivar has the highest stomatal density on abaxial side (253.00±29.05 stomata/mm2 ) and SU50 cultivar has the highest stomatal density on adaxial side (101.00±11.66 stomata/mm2). Silica cells occur abundant on abaxial side (101-123 cells/mm2) with dumb-bell shaped in all cultivars while KPS01-12 cultivar showed the highest number (126±31.14 silica cell/mm2). Cork cells present high density in KK3, UT84-12 and KPS01-12 cultivars (78.00±10.33, 77.00±16.39, 75.00±6.96 cells/mm2, respectively) on abaxial surface. Keywords: sugarcane, stomata, silica cell, cork cell P-023 Characteristic, distribution and density of stomata, silica cell and cork cell in 5 cultivars of sugarcane (Saccharum officinarum L.) REFERENCES [1] Ivan, A.R. (2005). Medicinal Plants of the World Volume 3 Chemical Constituents, Traditional and Modern Medicinal Uses. New York: Humana Press. [2] Paopun, Y., Thanomchat, P. and Toyen, D. (2022). Analysis of biosilica in sugarcane leaves. Microscopy and Microanalysis Research, 35(2), pp. 5-9. [3] Worasitikulya, T., Thapakor, R., Nuntawoot, J., Sayam, R. and Pitaakpong, M. (2019). Leaf anatomical responses to drought stress condition in hybrid sugarcane leaf. Malaysian Applied Biology, 48(3), pp. 181-188.

127 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 Siriporn Donnua1 *, Niphone Thaveechai², Prakai Ratchanu-Wong², Udomsak Lertsuchatavanich², and Thamrongjet Pattamuk⁵ 1 Department of Plant Pathology, Faculty of Agriculture at Khampkhaeng Saen, Kasetsart University, Nakhon Pathom, 73140, Thailand ²Retired ³Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand ⁴Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand ⁵Major of Plant Production Management and Urban Agriculture Learning Center, Department of Agriculture and Cooperatives, Sukhothai Thammathirat Open University, Nonthaburi, 11120, Thailand *Correspondence to: Department of Plant Pathology, Faculty of Agriculture at Khampkhaeng Saen, Kasetsart University, Nakhon Pathom, 73140, Thailand. E-mail; [email protected] ABSTRACT: A sugarcane screening model for sugarcane white leaf (SCWL) disease-resistant varieties was developed and tested for disease-resistant screening of seedlings from cuttings and of seedlings from tissue culture. Three varieties of sugarcane were tested: KK3 (susceptible), LK92 (moderately tolerant) and UT15 (tolerant). Phytoplasma was inoculated into sugarcane seedlings by an insect vector, Matsumuratettix hiroglyphicus Matsumura in plastic cages. Resistance evaluation was investigated based on symptom evaluation and phytoplasma quantification using TaqMan real-time polymerase chain reaction (PCR). Results found that the KK3 variety was the most susceptible which showed white leaf on ratoons at fifth to sixth month after inoculated when neither nor in other two varieties (LK92 and UT15). Phytoplasma quantification by TaqMan real-time PCR showed that the KK3 variety had a lower phytoplasma titer than either the LK92 or UT15 varieties, respectively. This experiment indicated that the UT15 variety was more tolerant to white leaf disease than the other two varieties, despite having the highest titer of phytoplasma but did not present any white leaf symptoms. The KK3 variety was the most susceptible, showing white leaf with the lowest titer. In screening for sugarcane white leaf disease resistance, the recommended approach is to use KK3 for a susceptible check and UT15 for a resistant or tolerant check. The screening model from this experiment could be applied in primary screening for SCWL disease-resistant varieties on seedlings from seeds grown-out, plants from tissue culture or seedlings from cuttings. This model is useful for rapid and accurate screening of SCWL disease resistance. Keyword: sugarcane; resistant variety; model; white leaf disease; phytoplasma. P-024 Sugarcane screening model for sugarcane varieties resistant to white leaf disease

128 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 white leaf disease (SCWL) caused by Candidatus Phytoplasma; 16rXI [1]. The SCWL disease effects huge economic losses to sugar and cane industries in Thailand. Infected sugarcanes produce low sugar content and low productivity [1, 2, 3]. Severely infected fields had 100% yield losses [4]. The SCWL phytoplasma is transmitted by two main planthoppers; Matsumuratettix hiroglyphicus Matsumura and Yamatotetix flavovitatus Matsumura [5]. The most serious epidemic manner is via planting materials or cuttings. Hot water and tetracycline treatment of cuttings, planting of disease-free plants, sanitation, crop rotation, insecticide application, regulatory quarantine, were introduced to SCWL disease management [1, 6, 7]. Disease control should follow good cultural practices. Using resistant variety is the most effective method to control disease but non sugarcane variety which resistance to SCWL disease was introduced to sugarcane production. The screening for SCWL disease-resistant variety model was developed and validated on seedlings from tissue culture, and seedlings from cuttings. This model is useful for rapid and accurate screening of SCWL resistance in future. 2. MATERIALS AND METHODS 3.1 Sugarcane seedlings One-month seedlings from cuttings were used in experiment I, and three-month seedlings from tissue culture were used in experiment II. Sugarcane seedlings of each treatment were grown in 32 meshes cages separately in each cage. Three varieties of sugarcane were tested; the KK3 was a represent susceptible, LK92 a represent moderately tolerant, and UT15 a represent tolerant. 3.2 Insect vector The planthopper; Matsumuratettix hiroglyphicus (Matsumura) were fed and multiplied on healthy sugarcane seedlings, the KK3 variety in plastic cages. The plastic cages were kept in 32 meshes nylon cages. Adult planthoppers were acquired on SCWL-infected sugarcane in plastic cage for two days then planthoppers were transferred to new plastic cages with healthy sugarcane seedlings, for phytoplasma incubation within infested insects. This incubation period of phytoplasma in insect vector was taken for 20-days before used in inoculation. 3.3 Phytoplasma inoculation using insect vector The experiments were divided into two categories, investigated in one-month old seedlings from cuttings (experiment I), and in three-month old seedlings from tissue culture (experiment II). Three varieties of sugarcane were tested on both experiments: the KK3 (a represent susceptible), LK92 (a represent moderately susceptible), and UT15 (a represent tolerant). In total 180 seedlings, each of three varieties composed of ten seedlings for inoculation test and ten seedlings for non-inoculation test (control). Three replicates of each treatment were tested. Each seedling was grown in separate plastic bags and kept in 32 meshes nylon cages randomly for inoculation. Inoculation in experiment I, one plastic cage composed of three seedlings of the KK3, LK92, and UT15. Five insects were inoculation feeding randomly in one cage for five days. Seedlings were transferred to the 32 meshes nylon cage and grown for four months, then sugarcanes were cut-off and wait for shoot proliferation from ratoon. White leaf disease was evaluated on ratoons by symptom appearance and PCR detection of phytoplasma pathogen. Inoculation in experiment II, one plastic cage composed of ten seedlings of the KK3, LK92, and UT15. Ten insects were inoculation feeding randomly in one cage for one week. Ten seedlings of each variety were divided into two groups, five seedlings were grown in rice husk ash and another five seedlings were grown in loamy soil, in plastic bags and kept in 32 meshes nylon cage. White leaf symptoms were evaluated every week after inoculated. Phytoplasma was randomly detected by TaqMan real-time PCR every two weeks for ten weeks as described in 3.4. 3.4 Resistance evaluation The methods for resistant evaluation were symptom evaluation, white leaf symptoms in all seedlings were recorded every week for nine weeks, and quantification of phytoplasma by real-time PCR every two weeks for ten weeks. TaqMan real-time PCR was used to quantify secA gene of phytoplasma, the protocol as follows. Total genomic DNA was extracted from sugarcane midrib from all treatments by modified CTAB method [8, 9]. Quantifications of phytoplasma in all samples were detected by using TaqMan real-time PCR, used specific probe and primers to secA gene of the SCWL phytoplasma. The secA DNA target was 275 bp. The hydrolysis probe was labeled with FAM (6-crboxy-fluorescein) at 5’-end and TAMRA (6-carboxy-tetra-methyl-rhodomine) was labeled at 3’-end. The SCWL phytoplasma-specific primers and probe were SecAF(5’-GAAGCTAATAGTATTGAATTGA-3’),SecAR(5’-CCTGTAAATTGATCTATTATCAAA-3’), and secA probe(5’-AGAAGAAGGGATAAAGAAAGGTGAAA-3’). The TaqMan real-time PCR reactions using QuantiNova Probe PCR Kit (QIAGEN, CA., U.S.A.) were prepared in total 20 ul/ reaction composed of 10 ul 2xQuantiNova Probe PCR mix, 1µl DNA template,

129 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 0.5 µl 10uM SecAF forward primer, 0.5 µl 10uM SecAR reverse primer, and 0.2 ul 10uM secA probe. Total volume was adjusted with dH2O to 20 ul. Real-time PCR was run in the MyGO Mini PCR systems (IT-IS Life Science Ltd., Ireland) using program as followed: pre-incubation at 95 degree Celsius for 10 minutes; 45 cycles of amplification step by denaturation at 95 degree Celsius for 10 seconds, annealing at 60 degree Celsius for 30 second, and extension at 72 degree Celsius for 10 minutes. Phytoplasma concentrations in all samples were quantified by calculating using MyGO Mini software (v3.4.8) which compared to SP4 DNA standard. The SP4 DNA plasmid, recombinant plasmid of secA gene of the SCWL phytoplasma which was ligated into pGEM®- T Easy Vector (Promega, Madison, U.S.A.) from previous study used as DNA standard control. The DNA standard was investigated by ten-fold serial dilutions of the SP4 plasmid from 10-1-10-12 then measured by NanoDropTM 8000 Spectrophotometer (Thermo Scientific, U.S.A.) before quantified of secA gene by TaqMan real-time PCR. 3. RESULTS AND DISCUSSION The sugarcane screening model for SCWL disease resistant varieties was developed (Figure 1). The insect vectors; Matsumuratettix hiroglyphicus (Matsumura) (Figure 2a) were collected from Nakhon Sawan province, Thailand. Insect vectors were multiplied in 32 meshes nylon cages (Figure 2b), then were transferred for acquisition feeding on SCWL-infected plant (Figure 2c). Inoculation feeding on one-month seedlingsfrom cuttingsin experiment I (Figure 2d), inoculation feeding on three-month seedlings from tissue culture in experiment II (Figure 2e), both were taken for seven days in plastic cages. In experiment I, seedlings were grown in 32 meshes nylon cage after inoculated (Figure 3a). There were no white leaf symptoms on the two of each variety (KK3, UT15, and LK92) that had been inoculated compared to SCWL-infected leave (positive control); however, these treatment leaves were not dark green as those on healthy plants (negative control), as shown in Figure 3b. In ratoon stage (Figure 3c), ratoons of un-inoculated of all varieties, and inoculated ratoons of UT15 and LK92 did not showed white leaf. Only shoots of the KK3 ratoon showed white leaf (Figure 3d). In experiment II, seedlings of un-inoculated and inoculated of KK3, UT15, and LK92 varieties which were grown in loamy soil after inoculated for six weeks were almost similar (Figure 3e). Un-inoculated and inoculated seedlings which were grown in rice husk ash showed more stunt than in loamy soil, inoculated seedlings showed more stunt than un-inoculated seedlings(Figure 3f). Leaves of inoculated of the KK3 showed more white leaf than the LK92, and UT15 but less severe than SCWL-infected leaf, when healthy sugarcane leaf showed dark green (Figure 3g). Quantification of phytoplasma by using TaqMan real time PCR of secA gene at 4 weeks after inoculation in Table 1, inoculated samples showed 50-100x copies of phytoplasma more than un-inoculated samples. Phytoplasma copy number of inoculated samples of the KK3 variety increased to 5.64x104 copies when compared to un-inoculated sample, which had the lowest increased number, followed by the LK92, and UT15 to 3.97x105 , and 7.35x105 , respectively. This experiment indicated that UT15 variety was the most tolerant to white leaf disease than other two varieties even had the highest titer of phytoplasma but not show white leaf. The KK3 variety was the most susceptible which showed white leaf on ratoons with lowest titer of phytoplasma. Report of UT variety had two potential candidate protein biomarkers for reduced susceptibility to the SCWL disease were identified as proteins detected only in UT1 but not found in KK3 [10]. In screening for SCWL disease resistance probably use the KK3 for a susceptible check and UT15 for a resistant or tolerant check. Figure 1 The sugarcane screening model for sugarcane white leaf disease resistant varieties

130 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 v Figure 2 Insect vectors and inoculation feeding on seedlings in plastic cages; a) the insect vectors; Matsumuratettix hiroglyphicus (Matsumura) trapping, b) feeding of insects in nylon cage, c) acquisition feeding on SCWL-infected sugarcane, d.) five insects were fed on three seedlings from cuttings in one cage (experiment I), and e.) ten insects were fed on ten seedlings from tissue culture in each plastic cage (experiment II).

131 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 3 Sugarcane symptom in experiment I (a.-d.) and experiment II (e.-g.); a) seedlings were grown in 32 meshes nylon cage after inoculated, b) leaves from left to right, 1= healthy leaf (negative control), 2= SCWL-infected leaf (positive control), 3 and 4 = KK3 inoculated leaves, 5 and 6 UT15 = inoculated leaves, and 7 and 8 = LK92 leaves, c) ratoons of un-inoculated of all varieties, and inoculated ratoons of UT15, and LK92 which not showed white leaf symptom, d) white leaf symptom on shoot of KK3 variety after germinated from ratoon, e.) seedlings after inoculated for six weeks of KK3, UT15, and LK92 varieties compared to un-inoculated seedlings which were grown in loamy soil, f) inoculated and un-inoculated seedlings were grown in rice husk ash, and g) inoculated KK3, UT15, and LK92 leaves compared to SCWL infected and healthy sugarcane leaves.

132 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 Quantification of phytoplasma by using TaqMan real time PCR at 4 weeks after inoculated in experiment II. a Cut-off at 6,000 copies 4. CONCLUSIONS The sugarcane screening model for SCWL disease-resistant varieties was developed to be applied in primary screening for SCWL disease-resistant varieties of seedlings from seeds grown-out, of seedlings from tissue culture or seedlings from cuttings. Quantification of phytoplasma by using TaqMan real-time PCR using SCWL-specific probe and primers is helpful in resistance selection not only screening by symptom evaluation. The KK3 variety is suitable for a susceptible check and UT15 for a resistant or tolerant check. 5. ACKNOWLEDGEMENT This research was financially supported from The Thailand Research Fund (TRF) under the project code; RDG60T0198. 6. REFERENCES [1] Wongkaew P. (2012) Sugarcane white leaf disease characterization, diagnosis development, and control strategies. Functional Plant Science and Biotechnology, 6(Special Issue 2), pp. 73-84. [2] Hanboonsong Y., Ritthison W., Choosai C., Sirithrorn P. (2016). Transmission of sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector. Journal of Economic Entomology, 99, pp. 1531-1537. [3] Donnua S., Moonjuntha P. and Tiwari A.K. (2023). Diversity, distribution, and status of phytoplasma disease in Thailand. In A.K. Tiwari, K. Caglayan, A.M. Al-Sadi, M. Azadvar and S. Abeysinghe, (Eds), Phytoplasma Disease in Asian Countries. AP Academic Press. 31-38. [4] Rao G.P., Srivastava S., Gupta P.S., Sharma S.R., Singh A., Singh S., Singh M. and Marcone C. (2008). Detection of sugarcane grassy shoot phytoplasma infecting sugarcane in India and its phylogenetic relationships to closely related phytoplasma. Sugar Tech, 10(1), pp. 74-80. [5] 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 Molecular Biology, 11, pp. 97-103. [6] Hanboonsong Y. (2016). Insect Vector of Sugarcane White Leaf Disease and Their Management (in Thai). KKU printing. Mueang Khon Kaen District, Khon Kaen, Thailand. [7] Klinkong S. (2009). Plant Pathogenic Phytoplasma (in Thai). Petchrung Print Center, Mueang Nonthaburi District, Nonthaburi, Thailand. [8] Murray M.G. and Thompson W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, pp. 4321-4325. [9] Donnua S., Paradornuwat A., Chowpongpang S. and Thaveechai N. (2012). Comparison between single and duplex conventional PCR for detection of CandidatusLiberibacterasiaticus, the causal agent of citrus Huanglongbing disease in Thailand. Crop Protection, 41, pp. 128-133. [10] Leetanasaksakul K., Roytrakul S., Phaonakrop N., Kittisenachai S., Thaisakun S., Srithuanok N., Sriroth K., Soulard L. (2022). Discovery of potential protein biomarkers associated with sugarcane white leaf disease susceptibility using a comparative proteomic approach. PeerJ, 10:e12740 http://doi.org/10.7717/peerj.12740.

133 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 Thanankorn Jaiphong1 *, Titinai Thienyaem1 , Pisit Intarawirat1 and Nitirong Pongpanich1 ¹National Agricultural Machinery Center, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand Correspondence to: National Agricultural Machinery Center, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand. E-mail: [email protected] ABSTRACT: The sugarcane growers became popular using single bud cutting to make seedlings before transplanting to the field for many reasons as (1) to reduce the number of cane setts by approximately 80% for cultivation, (2) to a high survival rate, and (3) convenience for pre-treatment to disinfection of diseases and insects. However, cutting the single bud is done manually resulting in a lack of skilled workers and high labor costs. Thus, using the machine may reduce labor problems. The research aims to study, design, create, test, and evaluate the sugarcane-bud cutting machine and evaluation the germination of a single bud. The cutting machine uses a machine vision system for searching the cane buds and cut them to 3.50 cm. The results revealed bud searching system can be processed at 30 frames per second (FPS). High efficiency of searching was found at the speed of 27.77 cm/min with the ability to cut in the range of 600-700 buds/ hour. The system test found that the cutter could cut correctly at 90.41%, uncut at 8.63%, cut on the bud at 0.96%, and wrong cut at 0.55%. The comparison between cutting by machine and manual revealed the ability to cut at 635.70 and 473.30 buds/hour respectively or 34.31% higher by cutting machine. The results of germination showed the bud that was cut by machine was higher than by manual cutting at 5.60% and 28.10% in U-thong 17 and Khon Kaen 3 varieties, respectively at 35 days after seeding. In conclusion, the sugarcane-bud cutting machine reduces working time and labor costs. The quality of the bud results in good germination. Moreover, this machine has the potential to develop for high efficiency in the future. Keywords: Cutting machine, Machine vision system, Single bud, Sugarcane bud P-025 Sugarcane-Bud Cutting Machine

134 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 Patcharin Songsri1,2,*, Thanakorn Kulrat1 , Darunee Puangbut3 , Patcharee Suriya⁴, Panatda Utaranakorn⁴, Anucha Laoken5 and Nakorn Jongrungklang1,2 1 Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center (NECS), Khon Kaen University, Khon Kaen, 40002, Thailand ³Plant Production Technology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani, 41000, Thailand ⁴Department of Agricultural Economics, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand ⁵Mahasarakham Agricultural Research and Development Centre, Mueang, Mahasarakham, 44000, Thailand *Correspondence to: [email protected] ABSTRACT: The use of sugarcane harvester can reduce PM 2.5 caused by burning sugarcane before harvest and increase soil fertility from sugarcane leaves. Cane yield and nutrient volume in leaves may be affected by different genotypes and environmental conditions. Thus, the objective of this study was to determine cane yield and N, P, K content in the leaf of 5 sugarcane varieties harvested by sugarcane harvester in 3 experiment locations. The 5 sugarcane varieties (UT13, KK3, Kps01-12, KKU99-02 and KKU99-03) were planted under rainfed conditions in a randomized complete block design with four replications at 3 locations in Udon Thani, Mahasarakham and Khon Kaen Province from December 2022 to January 2023. The plot size had 4 rows 40 m long and a spacing of 1.65 m between rows. At harvest cane yield was harvested by sugarcane harvester and the data of cane yield, leaves dry matter, and nutrient contents (N, P, K) were recorded. Sugarcane varieties were significantly different for cane yield and leaves dry matter at two locations (Mahasarakham and Khon Kaen Province) and the variety, Kps01-12 had high both of cane yield and leaves dry matter. Whereas the KK3 and KKU99-02 had high leaves dry matter at Mahasarakham and Khon Kaen Province. The N and P volumes in leaves (kg/rai) were significantly different at three locations. Whilst K volume in leaves was significantly different at Mahasarakham and Khon Kaen Province. The variety, Kps01-12 and KKU99-02 had high N volume in leaves (6.45 – 9.22 kg/ rai) at all 3 locations and KK3 had high at Khon Kaen and Mahasarakham Province (8.12 and 9.25 kg/rai, respectively). P volume in leaves, Kps01-12 and KKU99-03 had high (1.09 -1.52 kg/rai) at Khon Kaen and Mahasarakham Province. KK3 had a high P volume in leaves 1.48 and 1.50 kg/rai, respectively at Khon Kaen and Udon Thani Province. K volume in leaves, KKU99-02 had high at Khon Kaen and Mahasarakham Province (12.58 and 9.81 kg/rai, respectively). Keywords: Saccharum spp, sugar cane leaf yield, residue, nutrients volume in leaf, PM 2.5 P-027 Evaluation of Cane Yield and N, P, K Content in Leaf of 5 Sugarcane Varieties Harvested by Sugarcane Harvester

135 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 Thanakorn Kulrat¹, Nakorn Jongrungklang1,2, Sanun Jogloy1,2, Amarawan Tippayawat1,3 and Patcharin Songsri1,2,* 1 Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center (NECS), Khon Kaen University, Khon Kaen, 40002, Thailand ³Khon Kaen Field Crops Research Center, Muang, Khon Kaen, 40000, Thailand *Correspondence to: [email protected] ABSTRACT: More than 80 percent of sugarcane plantations in Thailand were grown under rainfed conditions, and droughtstress normally affectsthe early growth stage.Interspecific sugarcane hybrids mostly crossed from Saccharum officianarum and Saccharum spontaneum for many breeding objectives, such as multipurpose cane (both sugar and energy), improving drought resistance level, and increasing nitrogen (N) use efficiency. Understanding N use efficiency (NUE) in different sugarcane genotypes is important for sugarcane breeding programs to improve yields under drought conditions. Thus, the objective of this study was to determine the NUE and cane yield of interspecific hybrids and commercial sugarcane genotypes grown under early drought conditions. The experiment was conducted under field conditions at Khon Kaen Field Crops Research Center of Tha Phra Campus, Khon Kaen Province, Thailand, from November 2020 to December 2021. The experiment was arranged in a split plot in a randomized complete block design with four replications. The main plot was represented by three drought durations: no water stress (WW), early drought stress (Rainfed; DS), whereas the subplot consisted of six sugarcane clones/varieties consisted of 4 interspecific hybrids (F03-362 (F1), KK99-0358 (BC1), TPJ04-768 (BC1) and KK99-0930 (BC2)) and 2 commercial varieties (KK3 (drought adaptive) and UT12 (drought susceptible)). Plot size was 12 m × 13 m with a spacing of 1.5 m between rows and 0.5 m between plants within a row. Biomass, N content (%), N uptake (kg/rai), and NUE (g/g) were measured at 6 months after planting (MAP) (drought stress period) and 12 MAP (recovery and harvest). Cane yield was measured at 12 MAP. At drought stress (6 MAP), biomass, N uptake, and NUE were significantly different in water regimes and genotypes (p≤0.01), except N content was significantly different in water regimes (p≤0.01). At 12 MAP, water regimes were highly significant difference (p≤0.01) in biomass and NUE, while N uptake was significantly different at p≤0.05. During the drought stress period (6 MAP), interspecific hybrids genotypes showed higher biomass and NUE than those commercial cane, especially F03-362 (F1). At harvest (12 MAP), KK3 had high biomass and NUE, both of which WW and DS conditions, and high cane yield under DS equal to interspecific hybrids genotypes (F03-362 (F1)). Key Words: S. officianarum, S. spontaneum, NUE, Nitrogen uptake, water stress P-028 Nitrogen use Efficiency and Cane Yield of Interspecific Hybrids and Commercial Sugarcane Genotypes Grown under Early Drought Conditions

136 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 Rungtip Ngaklunchon¹, Nakorn Jongrungklang1,2, Kittipat Ukoskit³, Collins Kimbeng⁴, Marvellous Zhou⁵, and Patcharin Songsri1,2,* ¹Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen Thailand 40002 ²Northeast Thailand Cane and Sugar Research Center, Khon Kaen University, Khon Kaen Thailand 40002 ³Department of Biotechnology, Thammasat University, (Rangsit Campus), Pathum Thani Thailand 12121 ⁴Department, Sugar Research Station, Louisiana State University (LSU), Baton Rouge, LA 70803, United States ⁵South African Sugarcane Research Institute, P. Bag Xo2, MT Edgecombe, 4300, South African Correspondence to: Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand. [email protected] ABSTRACT: Poor families indicate poor recovery of high-value selections from selection, implying that a considerably larger number of seedlings from poor families must be planted. Planting a greater number of seedlings is costly and will raise the cost of a breeding operation while yielding minimal genetic advances. The objectives of this study were to determine the genetic variability among interspecific crosses and progenies from the crosses and determine the potential of family evaluation to enhance the efficiency of selection. In 16 commercial traits from 46 interspecific hybrids progenies created by six crosses between three cultivars (UT5, F152, and KK07-559) and five wild species (THS98-41, THS98-91, THS98-95, THS98-94, and THS97-51), resulted in three crops were evaluated for genetic variability among crosses and progenies within crosses. The results showed differences in trait combinations among families suggesting possible selection of families with higher trait combinations. Families F1M2, F1M3 and F3M2 produced better trait combinations than families F1M1, F2M4 and F2M5, further highlighting the potential to select families with better trait combinations. Family F2M5 can be classified as an average family. Despite the small population of parents, there is an opportunity to identify the top families from where elite progenies can be selected. The values of family evaluation and selection demonstrated here suggest that evaluating and selecting elite families and selecting progenies from the best families reduce costs because smaller numbers will produce better results compared to larger numbers from poor families. Keyword: S. officinarum, S. spontaneum, Interspecific hybrid, Blup analysis, Sugarcane breeding P-029 Family and Progeny Genetic Variability and Family Evaluation for Fiber, CCS, Cane Yield and its Components Traits Derived from Interspecific Crosses

137 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 Pornthiwa Khwanthaworn1*, Patcharin Songsri1,2, and Nakorn Jongrungklang1,2* 1 Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen Thailand 40002 ²Northeast Thailand Cane and Sugar Research Center, Khon Kaen University, Khon Kaen Thailand 40002 *Correspondence to: [email protected] ABSTRACT: Sugarcane (Saccharum spp.), an important crop for both sugar and bioenergy production, is cultivated in tropical regions. Almost sugarcane in this region is produced under rain-fed conditions that often face drought stress situations. Currently, multipurpose and biomass canes become an important approach in the breeding aspect. However, biomass sampling is limited in some early-generation selections. The surrogate traits which represent biomass performance need to be established, especially non-destruction samples. Therefore, the objective of this study was to determine the agronomic and physiological traits that contributed to the biomass of a diverse set of sugarcane genotypes under different durations of drought. The experiment was conducted under field condition and arranged in a split plot in a randomized complete block design with four replications. The main plot was represented by three drought durations: no water stress (FC), short-term drought (SD), and long-term drought (LD), whereas the sub-plot consisted of six sugarcane genotypes. Agronomic traits and physiological traits were collected at 3, 6, 8, 10, and 12 months after transplanting (MAT). At 3 MAT, a positive correlation existed between canopy height and biomass in both FC and LD conditions, and between green leaves number and biomass in both FC and LD conditions. In addition, there was also found relationship between the number of tillers and biomass at 3 MAT under LD treatment. Non-destructive leaf area index was a trait that contributed to biomass at 6 MAT under non-water stress condition. At the physiological maturity stage, at 8 MAT, the positive relationship between canopy height and biomass was found under SD and LD conditions, and green leaves number also related biomass under FC conditions. Biomass at the harvesting stage was contributed from canopy width in sugarcanes that experienced FC conditions. Non-destructive traits in this experiment such as canopy height and number of green leaves could be used as indirect measurements to reflect the biomass performance under FC and LD conditions at tillering phase and at the physiological maturity phase. For the elongation phase, leaf area index that was observed as the non-destructive method was an altered character that determines biomass as indirect. This information will provide useful as an alternative measurement to indicate biomass in the breeding program of drought resistance at the early growth stage. Keyword: non-destructive, water deficit, leaf area index, inter-specific hybrid, and rainfed P-030 Contribution of agronomic and physiological traits to biomass of a divers set of sugarcane genotypes under early season drought.

138 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 Jidapa Khonghintaisong¹, Nakorn Jongrungklang1,2*, Patcharin Songsri1,2 ¹Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Correspondence to: [email protected] ABSTRACT: In tropical regions, water stress at the early growth stage of sugarcane is a usual occurrence around 2-4 months. Enhancing sugarcane production in drought-affected regionsis a key strategy, especially with the use of drought-resistant cultivars. However, the mechanisms and responses of physiological traits related to sugarcane drought resistance must first be understood, so that drought-resistant cultivars can be developed effectively. Therefore, the purpose of this research was to investigate the responses of physiological characteristics under early drought conditions of different drought-resistant levels of sugarcane cultivars and during the re-watering period. The experiment was conducted under pot conditions in a greenhouse. A 2×2 factorial in a completely randomized design (CRD) with four replications was used. Two waterregime management: field capacity (FC) and drought of early growth stage treatments(DE)(water withholding during 90-150 days after planting (DAP), after that re-watering period) were assigned as factor A. Sugarcane cultivars differing in water deficit-tolerant levels namely KK3 and UT12 were assigned as factor B. Physiological traits involved with photosynthesis namely Stomatal conductance (gs), transpiration rate (E), net photosynthetic rate (PN), water use efficiency (WUE), Photosystem II photochemistry under natural (Fvʹ/Fmʹ) condition, maximum quantum efficiency of photosystem II photochemistry (Fv/Fm) were measured at the 1st, 3rd, 5th, and 7th days after withholding (DAWW), 1 and 2 months after water withholding (MAWW), and also the 1st, 3rd, 5th, and 7th days after re-watering (DARW). During drought at the formative growth stage, sugarcane drought-resistant cultivar acclimated to drought as reached a maximum value in gs, E, PN, and WUE before midday, and gradually decreased then until sunset. The mechanism of the drought-resistant cultivar had a rapid reduction in gs, E, and PN but kept consistent with Fv/Fm for survival. However, drought resistance, the PN trait revealed a slow recovery at the 7th DARW, but rapid recoveries were found in Fv’/Fm’, Fv/Fm and WUE and some higher than well-water treatment. As a result, physiological traits in this recovery phase (grand growth period) may be key for determining further growth and yield. This knowledge would be useful in explaining appropriate behavior and recommending surrogate traits for improving sugarcane cultivars in breeding programs for drought avoidance at the early growth stage. Keyword: Net photosynthesis diurnal, Photochemical system II, Susceptible sugarcane cultivars, re-watering period, P-031 Physiological responses during drought at formative growth stage of sugarcane and recovery phase

139 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 Kanlayanee Wiangwiset¹, Patcharin Songsri1,2, Narumol Piwpuan3 , Nakorn Jongrungklang1,2* 1 Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ³Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai, Thailand *Correspondence to: [email protected] ABSTRACT: : Leaf anatomical traits are important adaptive traits that determine a crop's ability to thrive in water-limited environments. Inter-specific hybridization with a wild cane could transfer drought-resistant characteristics that are limited in commercial cane, however, the understanding of the diversity and distribution of the offspring derived from inter-specific hybridization needs to be established first. Thus, the objective of this study was to assess the diversity of anatomical traits in the sugarcane interspecific hybrid backcross 1 (BC1) population. A backcross population was conducted as crossed between female (Saccharum spp. hybrid) and male (F1 inter-specific hybrid) parents, between UT5 (commercial cane) x F4-19 (interspecific hybrid). Anatomical leaf characteristics were measured at 7 months after planting: leaf thickness (μm), cuticle thickness (μm), the percentage of cuticle thickness (%), the percentage of bulliform cell vertical length (%), and the percentage of stomatal crypt depth (%). BC1 hybrid clones differed in leaf thickness, cuticle thickness, the percentage of cuticle thickness, the percentage of bulliform cell vertical length, and the percentage of stomatal crypt depth and were distributed between male and female parents. In terms of heterosis, there were 6 clones that revealed positive heterosis values in the percentage of stomatal crypt depth and had remarkably high heterosis. Seventy-five ‘BC1-1’ clones were spread evenly for cuticle thickness percentage, including 22 clones (30% of 73 ‘BC1-1’ progenies) that have a value for cuticle thickness percentage between 2.96–3.50%, heights higher than the male parent (heterosis ranged from 10.83%), and 13 clones between female and mid-parent. However, some ‘BC1-1’ clones had higher percentages of bulliform cell heights than the female parent 20 clones (27% of 73 ‘BC1-1’ progenies), which has a value between 31.5–43.4%, whereas ‘BC1-1’ hybrids (21.6–29.06%) had a lower value than the male parent, and the remaining clones had a distribution between the female and male parents (heterosis ranged 0.43%). Apparently, the stomatal crypt depth percentages of 73 ‘BC1-1’ progenies were higher than mid-parent values and distributed between the mid-parent and the male genotype. This information can support further sugarcane varietal improvement for resisting drought in the inter-specific hybridization breeding program. Keyword: Backcross progenies, Cross-section, Cuticle thickness, Wild cane, Stomatal crypt depth P-032 Diversity of leaf anatomy involves with transpiration in sugarcane backcross 1 (BC1) derived from interspecific hybridization

140 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 Sujittra Gongka¹, Sompong Chankaew¹, Tidarat Monkham¹, Nakorn Jongrungklang1,2 and Santimaitree Gonkhamdee1,2,* ¹Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Correspondence to: [email protected] ABSTRACT: Weeds are important pests which reduce the quality and quantity of sugarcane production in Thailand.The diversity and abundance of weeds or dominant weeds can have an impact on the field,resulting in a decrease in the effectiveness of commonly used weed control applications, which is the main cause of sugarcane yield loss. The objective of this study was to evaluate the effectiveness of weed control methods on some of the dominant weeds in sugarcane production in North-East Thailand which conducted ten weed control applications in randomized complete block design (RCBD) with four replications, the sugarcane variety was conducted on KK3 which was planted in the late-rainy season in upland field 1 locations, December 2020 to December 18, 2021, Nong Han District, UdonThani Province; in addition, weed sampling to collected dominant weed species data which calculated the percentage of summed dominance weed ratio (SDR). We found a significantly differences of weed control applications in pre-emergence herbicide applications of pendimethalin + imazapic, indaziflam and indaziflam + sulfentrazone at recommend rate. The three treatments gave the highest weed control efficiency in the critical period of sugarcane, the efficiency was consistent with the low SDR values of the main weedsfound.The dominant weedsfound were Brachiaria distachya (L.) Stapf., Digitaria ciliaris (Retz.) Koel, Dactyloctenium aegyptium (L.) P. Beauv., Merremia cissoides Lam., Ipomoea gracilis R.Br. and Sida cordifolia L. However, the most problematic weed in the field was Brachiaria distachya (L.) Stapf. due to the highest inter-species SDR (72.94%), this may be related to the nature of the ability to germinate throughout the year. Thus, these findings indicate the use of a single pre-emergence herbicide is indaziflam at recommend rate, was sufficient to control only the dominant weeds in sugarcane fields as the mentioned above which soil type is Sandy Loam and the average rainfall throughout the experiment was 48 millimeters. In this regard, this study provides useful information for selecting weed control methods in sugarcane fields in North-East Thailand. Keyword: Main weeds, Dominant weeds, Critical period of sugarcane, Weed management, Unwanted flora P-033 Effect of pre-emergence herbicide on weed control of sugarcane plantations in Northeastern Thailand

141 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 Wishchabhas E-sa1,2,3 and Wanwipa Kaewpradit1,2,3* 1 Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand ²Northeast Thailand Cane and Sugar Research Center, Khon Kaen University, Khon Kaen, 40002, Thailand ³Applied engineering for important Crop of the Northeast Group, Khon Kaen University, Khon Kaen, 40002, Thailand *Correspondence to: Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand. E-mail: [email protected] ABSTRACT: Thailand, the world’s second-largest sugarcane producer, cultivates sugarcane over a million hectares and annually produces 75 million tons of sugar produced. The sugar production area in northeastern Thailand accounts for over 40% of the nation’s total sugarcane cultivation. A common practice is pre-harvest burning due to its efficiency in lowering labor requirements. However, such pre-harvest sugarcane burning contributes to particulate matter 2.5 (PM2.5) emissions and may influence changes in soil microbial biomass. This study aimed to investigate the alteration in soil microbial biomass carbon and nitrogen during ratoon cane harvest following a shift from consecutive burning to green cane harvest management. The experiment was conducted under sandy soil with low organic matter content. There were two treatments with four replications for ratoon cane: 1) pre-harvest burning and 2) green cane harvest. A comparison between treatments was performed using the T-test. Our result showed that transitioning to green cane harvest from consecutive burning management significantly increased microbial biomass carbon and nitrogen (7% and 193%, respectively). Thus, green sugarcane harvest may offer an environmentally friendly solution enhancing soil microbial biomass and supporting the sustainability of the sugarcane cropping system. Keyword: PM2.5, organic matter, soil C:N ratio P-034 Impact of transitioning from consecutive burning to green cane harvest on soil microbial biomass carbon and nitrogen in sandy soil conditions of Thailand

142 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 Khwantri Saengprachatanarug ¹Faculty of Engineering, Khon Kaen University, Thailand Correspondence to: [email protected] ABSTRACT: Efficient management systems require a good planning system that generates work plans based on a large amount of data and can adjust plans automatically in real-time to respond to situational changes or issues. Therefore, a planning system must work in conjunction with a monitoring system and be able to receive commands to execute new plans. The platform that operates in this network must cover everything from farm design, planting, cultivation, plant care, harvesting, transportation, and customization for each user or crop type. The platform requires input such as data, models, and basic technologies to function effectively. When the management system is efficient, it leads to a reduction in production costs in the agriculture industry. To meet the needs of the target industry group, as mentioned in the hypothesis of the aforementioned research, the service business will consist of three main modules that will use the platform developed each year. Each module has a service format as shown in Table 1. P-035 Precision Agriculture Platform for sugar-Industry Sustainability: A Field Practice Solution Precision Agriculture Platform for sugar-Industry Sustainability: A Field Practice Solution Khwantri Saengprachatanarug Faculty of Engineering, Khon Kaen University, Thailand *Corresponding Author [email protected] Abstract Efficient management systems require a good planning system that generates work plans based on a large amount of data and can adjust plans automatically in real-time to respond to situational changes or issues. Therefore, a planning system must work in conjunction with a monitoring system and be able to receive commands to execute new plans. The platform that operates in this network must cover everything from farm design, planting, cultivation, plant care, harvesting, transportation, and customization for each user or crop type. The platform requires input such as data, models, and basic technologies to function effectively. When the management system is efficient, it leads to a reduction in production costs in the agriculture industry. To meet the needs of the target industry group, as mentioned in the hypothesis of the aforementioned research, the service business will consist of three main modules that will use the platform developed each year. Each module has a service format as shown in Table 1. Table 1 Table caption. No. Module Services 1 Farm Monitoring and Mapping services (FMM) Provide aerial photography-based field condition assessment services using unmanned aerial vehicles, create reports and automatically record them as GIS databases. The assessment covers 4 aspects including (1) yield quantity (2) product quality (sweetness) (3) plant diseases (4) plant fertilizer requirements. 2 Farm Robotic Solution services (FRS) Provide design and installation services for automatic/semi-automatic control systems by developing AI for command ordering using data from crop monitoring in year 1 (Data to Action). The solution includes: 1) Mobile-KIT for in-field activity tracking and advisory 2) Variable rate spraying drone 3) Chemical automatic mixing machine for drone. 3 Farm Business Intelligent services (FBI) Provide an intelligent planning and automatic adjustment system based on current situations, using data from FMM service in year 1 and FRS service in year 2. The plan is divided into sub-modules that users can select to use, including: o Weed management plan with spot spraying system for agricultural drones o High-precision positioning UAV system to reduce operating costs related to image capture and processing for field assessment o Decision support system for field management, including plans for machinery maintenance and harvesting (Full Factory Capacity/ Max. quality/ Min. Cost)

143 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 development of farm monitoring and mapping system is the prototype of a semi-industrial technology level as well as creating the yield map, the plant disease outbreak map from UAV imagery and automatic geographic information systems (GIS). As a result, from flight data, the yield map, and the plant disease outbreak map were able to be computing photos covering at least 200 rai per 1 computing and can display resolutions from GSD 3 -15 cm.The appropriate size was GSD 8-10 cm and took 1 hour 17 minutes for computing 100 rai. When compared to the existing system, it takes at least 4 hours. The latest developed system takes about 3 times less time or 68% and operation cost is lower than using the program in the market 63%. Farm robotic solution focuses on the development of information management systems, and automatic/semi-automatic control systems by developing AI for commands using data to measure farm conditions from the farm monitoring and mapping system. Activities consisted of system design, prototype fabrication, testing, and the break-even point analysis. There were 3 main outputs; 1) Mobile-KIT for in-field activity tracking and advisory 2) Variable rate spraying drone 3) Chemical automatic mixing machine for drone. According to the test, all products were effective and passed the technical target values, and are ready to provide services to the first target group, the sugarcane and sugar industries. Farm business intelligent system consists of weed control solution, high-precision positioning UAV system and decision support system for field management. The key element in developing a processing model for a weed control solution is the development of AI for detecting and classifying types of weeds and AI for recommending management methods based on a large number of environmental conditions. This is related to building a database and presenting solutions to help users make smart decisions (a decision supportsystem). Thissystem istherefore a part that helps businesses orstakeholdersin agriculture, pesticide service providers, and raw material buyers (sugar mills) to make informed decisions and reduce the risk of errors and losses due to inadequate weed control. Refer to the experiment results, it was found that site-specific spraying during the tillering and sugar formation stages helped reduce the cost of chemicals by around 50-60%, and also allowed for efficient battery use. Additionally, using a drone for spraying can reduce the spraying cost by at least 30%. Fig. 1 Figure caption. The development of farm monitoring and mapping system is the prototype of a semi-industrial technology level as well as creating the yield map, the plant disease outbreak map from UAV imagery and automatic geographic information systems (GIS). As a result, from flight data, the yield map, and the plant disease outbreak map were able to be computing photos covering at least 200 rai per 1 computing and can display resolutions from GSD 3 -15 cm. The appropriate size was GSD 8-10 cm and took 1 hour 17 minutes for computing 100 rai. When compared to the existing system, it takes at least 4 hours. The latest developed system takes about 3 times less time or 68% and operation cost is lower than using the program in the market 63%. Farm robotic solution focuses on the development of information management systems, and automatic/semi-automatic control systems by developing AI for commands using data to measure farm conditions from the farm monitoring and mapping system. Activities consisted of system design, prototype fabrication, testing, and the break-even point analysis. There were 3 main outputs; 1) Mobile-KIT for in-field activity tracking and advisory 2) Variable rate spraying drone 3) Chemical automatic mixing machine for drone. According to the test, all products were effective and passed the technical target values, and are ready to provide services to the first target group, the sugarcane and sugar industries. Farm business intelligent system consists of weed control solution, high-precision positioning UAV system and decision support system for field management. The key element in developing a processing model for a weed control solution is the development of AI for detecting and classifying types of weeds and AI for recommending management methods based on a large number of environmental conditions. This is related to building a database and presenting solutions to help users make smart decisions (a decision support system). This system is therefore a part that helps businesses or stakeholders in agriculture, pesticide service providers, and raw material buyers (sugar mills) to make informed decisions and reduce the risk of errors and losses due to inadequate weed control. Refer to the experiment results, it was found that site-specific spraying during the tillering and sugar formation stages helped reduce the cost of chemicals by around 50-60%, and also allowed for efficient battery use. Additionally, using a drone for spraying can reduce the spraying cost by at least 30%. Machine Dashboard Fertilizer suggestion Phase 2 GIS-Map-Report Yield Brix Disease NPK Phase 1 Integration Optimization Optimization Integration Target group: Sugar industry Farmer contract system VETAL VESPA HEX HGMC HiveGrid HGMC HiveGrid VRT sprayer Mobile-KIT for in-field activity tracking Phase 3 Farm management decision support system • Harvest scheduling simulation • Activity management • PPK GPS module and firmware • Comunication system with base station High-precision positioning UAV system Weed control solution • Weed detection • Spraying suggestion • Flight planning and control for site-specific weed control Auto-mixing machine UAV testing standard and HR development model 2 1 3 4

144 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING

145 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Lerdej Kwangthong1 *, and Charles Frederick2 1 Sutech Consultant Co., Ltd., 17th Sinn Sathorn Tower 77/68 Klongtonsai, Klongsarn, Bangkok 10600 Thailand 2 Sutech Engineering Co., Ltd., 17th Sinn Sathorn Tower 77/64 Klongtonsai, Klongsarn, Bangkok 10600 Thailand *Correspondence to: [email protected], and [email protected] ABSTRACT: The use of Mechanical Vapour Recompression has been investigated in Cane Sugar factories for at least the last four decades. MVR generally utilizes electrically driven single stage centrifugal compressors and high-pressure fans to compress low grade vapors to achieve higher pressure (and temperature) vapors which can be effectively used for heating duties in a sugar factory. Typically, MVR’s are employed on juice evaporator trains to compress the downstream vapors for reuse in the upstream effects of the evaporator. Sugar factory installations which experience a below requirement process steam availability, and do not wish to consider the high Capital Expenditure of increasing their steam generation capacity, can consider the installation of MVR as a more cost effective option. In the case of the Factory where this study was executed the objective was to address a steam supply shortage of 20 to 30 tonnes per hour and achieve a reduction in bagasse usage, and not incur the expense of installing additional boiler capacity. To achieve this a two stage MVR unit was installed to compress the vapor exiting the 3rd effect Juice Evaporator body (V3) and reintroduce the vapour at a higher enthalpy (pressure & temperature) to combine with the V1 vapour exiting the 1st effect Juice Evaporator body, and by doing so reduce the exhaust steam TPH supply to the 1st effect Juice Evaporator body (steam demand). An associated effect of repurposing a portion of the V3 was the reduction in the final effect Juice Evaporator vapor feed to the condenser thus requiring a reduced quantity of injection water. This Case Study explains the objective of the MVR installation, overviews the installed equipment, provides a reviews and analyses the trials, and concludes with the results and recommendations. Keywords: MVR, V1 (vapour), V3 (vapour), Steam supply, Juice Evaporator, etc O-010 Mechanical Vapour Recompession (MVR) in a Philipino Sugar Factory A Case Study - Part 1

146 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING 1. INTRODUCTION The 7,500 Tons Cane per Day (TCD) Sugar factory was experiencing a shortage of process steam and required a solution without considering the high Capital Expenditure associated with the installation of extra boiler capacity. It was suggested that an MVR could be installed to utilize their existing steam generating capacity in conjunction with a desire to expand the factory’s cane milling rate to 8,000 TCD thereby leading to a more energy efficient installation. Overview of MVR Basics1 MVR’s use the principle of compressing steam, or vapour, to increase the enthalpy, temperature and pressure. Centrifugal compressors are a useful tool to perform the compression and in this case are driven by electrical motors. The electrical energy provided by the drive motor is transferred to the vapour as it passes through the centrifugal compressor by increasing its temperature and pressure and hence its thermal energy. Where the MVR was Installed The Factory dates from the 1950’s and has a quintuplet juice Evaporator consisting of a number of bodies which vary in type and age. After studying the process house it was decided that the most suitable location to install the MVR within the Juice Evaporator train to boost the temperature, and pressure, of the third effect evaporator vapour (V3) so that it could be combined with the first effect vapour (V1) thus effectively increasing the available V1 to perform it various heating duties in the process house. With the increase in the tons per hour of V1 available the evaporator bodies were reconfigured to optimize the available heating surfaces. Table 1 shows the duty allocation of the Juice Evaporator bodies before and after reconfiguration. To facilitate the reconfiguration of the Evaporator body’s changes to the vapour and juice pipelines were required. Most of this work was carried out during the milling season with the necessary ‘tie-ins’ made during the non-milling season. The MVR installation was connected to the Juice Evaporator with the necessary piping and valves to enable it to be by-passed at any given time. This also allowed the trials to take place with and without the operation of the MVR. Table 1 – Juice Evaporator

147 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Figure 2 – Process configuration of MVR installation in Juice Evaporator 2. INSTALLATION The MVR installation is a centrifugal compressorsystem with the two stagesinstalled in series. The compressors are supplied on separate skids with the piping fabricated on site and assembly of the remaining system components. Each compressor is directly driven by an electrical AC motor which are speed controlled by a Variable Frequency Drive device (VFD) and the system is controlled by a PLC. The system and compressor units are designed and supplied by Jiangsu JinTongLing Fluid Machinery Technology Co. Ltd of China. Figure 1 Diagrammatic configuration of the MVR system

148 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Table 2 - Specification details of the MVR units Control Strategy The system parameters are monitored by a number of field instruments that provide inputs to a PLC which controls the speed of the compressors via VFDs, and valved flow control devices. Each of the MVR stages can be set to maintain a constant vapour mass flow rate through the unit, kg/hr, or constant vapour discharge pressure, kPa, with this being achieved by modulating the rotational speed of the compressors. V3 is progressively compressed by each MVR stage to achieve the pressure equal to V1 in the header. A bypass across each compressor ensures balanced flow rates between both stages of the MVR. The outlet temperature of the vapour after each stage is maintained within the system design parameters with the injection of condensate as required. 3. DISCUSSION AND RESULTS The objective of this study was to address a steam supply shortage of 20 to 30 tons per hour by the Factory and achieve a reduction in bagasse usage, and not incur the expenditure associated with installing additional boiler capacity. In addition there was the desire to expand the milling rate of the factory to 8,000 TCD. The supply, installation of the equipment and modifications to the Evaporator took place during 2020 and the system was mechanically commissioned in October 2021. Initially the system was set to control the compressed vapour across both MVR stages and supply the V1 header at a constant mass flow rate however, due to the varying V1 consumption rate by the Batch Pan users, excess V1 was periodically experienced. For this reason the control point was reset to achieve constant vapour discharge pressure to match the pressure of the vapour exiting the 1st Juice Evaporator, but at a higher temperature. This proved successful in balancing the supply and vapour demand between V3 & V1. The Covid-19 pandemic detrimentally affected what could be achieved with this study. Although the system was mechanically commissioned with Sutech in attendance, Factory staff had to be relied upon for its operation and process commissioning. To add to this difficulty, the Chinese supplier of the MVR system could not visit the Factory site. As the system was new to the Factory operating personnel, they did their best to run it but with their priorities being on sugar production, using the inconsistent cane supply, their focus was not on the operation of the MVR system. It was operated on and off over differing production conditions with the daily Factory production reports analyzed, from which the below conclusions are made 4. CONCLUSION 4.1 The expected increase in cane supply to the factory was not realized which prevented the mill rate of 8,000 TCD being achieved, 4.2 Process make-up High Pressure (HP) steam to the Pressure Reducing & De-Superheater (PRDS) was significantly reduced, 4.3 Increased HP steam to the Turbo Generator (TG) was required to generate the electrical power required for the MVR compressors,