Outbreaks of Fusarium ear rot on Maize in Thailand

Outbreaks of Fusarium ear rot on Maize in Thailand

Warapon Bunkoed 1 Patcharavipa Chaijuckam 2 Tiyakhon Chatnaparat 2 Supot Kasam 2 Sodsai

Changsaluk 1, Jeeranan Yhamsoongnern 1 and Sutruedee Prathuangwong 2*

1 National Corn and Sorghum Research Center, Faculty of Agriculture, Pak Chong, Nakhon Ratchasima 30320, Thailand
2 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900,Thailand

*Corresponding authors; e-mail: [email protected]

ABSTRACT

A causal fungus of ear rot threaten to food security, most frequently isolated from infected
maize plants grown in 6 key locations around Thailand during 2016-2017, was identified as
Fusarium verticillioides based on cultural and morphological characteristics after the positive test for
fungal infection. The highest incidence of the disease was 100% in all locations surveyed with
severity ranged from 2-80%. Field trials by artificial inoculation; and natural incidence exhibited one
out of 60 inbred lines Ki30; and two-University (Kasetsart) varieties out of 20 tested, KSX 5720 and
KSX5911 possibly revealed resistance tendency that could be compared with those 3-commercial
varieties. More importantly, the high fumonisin on affected grains was detected in some location
samples with over the 5 ppm concentration levels using the direct competitive ELISA assay. This is
the first report of ear rot outbreak in the country that a comprehensive molecular characterization
needs to be further elucidates for F. verticillioides.

Keywords: Fusarium verticillioides, mycotoxin, ELISA, source of resistance

Introduction

Ear rots of maize occur worldwide wherever maize is grown (Kommedahl and Windels,
1981). Many fungal species infect maize grains including Fusarium spp., Aspergillus spp.,
Penicillium spp, and Stenocarpella spp. (Payne, 1999 a). Fusarium is one of the major fungal genera
associated with maize in Thailand (Darnetty and Salleh, 2013). This pathogen causes losses in grain
yield and quality, due to contamination of grains by mycotoxins, primarily fumonisin (Parsons and
Munkvold, 2012) Mycotoxins however, are presistant, thermostable metabolites that produce in
association with food and feeds, possibly cause health problems to human and animal (Sydenham et
al., 1991). Different species of fungi in the genus Fusarium can cause maize ear rot including
F.verticillioides, F. proliferatum, F. nygamai, and F. graminearum (Mukanga et al, 2010).The most
common species is F. verticillioides syn. F. moniliforme (Teleopmorph: Gibberella fujikuroi).
(Adejumo et al., 2007) F.verticillioides, and other Fusarium species that they are mostly overwinter in
infected crop debris (Cotton and Munkvold, 1998). Mycelium in infected crop debris produces
macroconidia and microconidia that is wind and rain splash disseminated, infecting ears through silks
and colonizing kernels. F. verticillioides can also infect maize plants systemically in which case ears
may be infected through the ear shank. Insects such as the European corn borer have also been
reported to act as vector and transfer F. verticillioides spores between plants or cause plant injury that
enable the fungi to infect the plant. Fusarium ear rot is characterized by cottony mycelium growth that
typically occurs on a few kernels or is limited to certain parts of the ear, unlike Gibberella ear rot.
Mycelium is generally white, pale pink or pale lavender. Infected kernels typically display white
streaking (also known as ‘starburst’ symptoms) on the pericarp and often germinate on the cob.
Typically, infection occurs close to ear tips and is commonly associated with damage and injury
caused by ear borers. Under severe infestation, the entire ear appears withered and is characterized by
mycelium growth between kernels (Payne, 1999a).

Management of ear rot of maize is very challenging. Foliar sprays with fungicides are not
effective and not economically feasible for maize grower in Thailand. Then integrating use of cultural
practices, seed treatment, and cultivation of resistance maize varieties possibly more effective to
manage the disease and reduce mycotoxin contamination in maize grains. Little research on this

disease was conducted in Thailand, disease incidence and severity, affected location, and the causal
fungus mainly Fusarium spp. were then, determined. Evaluation of host resistance for pathogen
infection of maize inbred lines and hybrid pre-commercial varieties and identification of mycotoxin
chemotypes of the causal Fusarium sp. from affected grains was also carried out in this study. Data
obtained may highlight the possibility of improving maize plant response to ear rot disease.

Materials and Methods

Sample collection

The occurrence of ear rot of maize was determined over a 2- year (2016-2017) growing
seasons (September to October) from fields in 6-key locations (6 districts) mainly cultivated for maize
production and suffered from this disease. Each sample collected from one randomly selected cob of
each hybrid variety and 23 crops were obtained from each location. In total, 138 samples were
collected from experimental fields in 6 districts. Sampling date and location (geographic coordinates)
were recorded from a global positioning system (GPS). Rainfall, relative humidity and temperature
data were collected during growing periods (August to October) obtained from the Department of
Meteorology website. Symptomatic cobs sampled were placed in plastic bags and delivered to
National Corn and Sorghum Research Center (NCSRC), Nakhon Ratchasima for further analysis.

Identification and isolation

The infected kernels of each cob sampled were surface-disinfected for 1 min in 3.5% NaOCl
then, rinsed twice in sterile distilled water. Thirteen maize kernels were plated on moist, sterile filter
paper in plastic Petri plates. The plated kernels were incubated at room temperature (25+2oC) for 7
days. For identification, some of the cultures were transferred to a 1/4-strength Potato Dextrose Agar
(PDA) medium for single spore isolation and then incubated for 7 days at room temperature (25+2oC).
The morphological and cultural characters, i.e. the pigmentation and the extent of mycelial growth,
shape and size of macroconidia, microconidia, nature of conidiogenous cells, the presence/absence of
macroconidia, chlamydospores and perithecia, were used to identify the species. These characteristics
were compared with those described previously (Barnett and Hunter, 1972; Mathur and Kongsdal,
2003).

Pathogenicity assay

Three corn varieties (WS4452, CP888 and NS3) were used in pathogenicity test. Conidial
inoculum of 6 Fusarium isolates that were identified as F. verticilioldes, was prepared following the
procedure of Zainudin et al. (2016) with minor modification. The concentration was modified to 2 ×
106 conidia/mL using a hemacytometer. After that, 1 mL of conidial suspension was injected to the
ear region using a sterile syringe at 70-day old plants. Two controls were set up, one inoculated with
sterile distilled water and one not inoculated. After 21 days of inoculation, the inoculated cobs were
manually dehusked and scored for the discoloration of kernels around the inoculation area. Evaluation
was done based on a disease scale from 1 to 7 as described by Reid and Hamiton (1996). To ascertain
the pathogenicity phenotypes of F. verticillioides, all inoculated ears showing Fusarium ear rot
symptoms were reisolated for single-spored, and reidentified based on their cultural and
morphological characteristics.

Maize inbred screening trial

A set of 60 inbred lines belonging to NCSRC, Kasetsart Univercity, Nakhon Ratchasima
province, was evaluated in rainy season (2017) under field trial at its resrarch station. with the aim
to find of maize resistance to Fusarium ear rot. Plots were arranged as randomized complete block
designs with three replications to evaluate plant resistance under artificial infection. The silk channel
inoculation (SILK) method described by Reid et al. (1993) where 2 ml of a macroconidial suspension
of F.verticilliodes at concentration of 105 conidia ml-1 was injected with blunt needle into the silk

channels of individual ears. Individual plots were 5 m long, 3 m wide and consisted in four rows
planted to 25 seeds per row. At maturity when kernel moisture was less than 20%, ears were manually
harvested and after hand de-husking, the field severity of F.verticilliodes infection was measured
using the disease rating of Reid and Hamiton (1996) as follows: 1=0% infection, 2=1-3%, 3=4-10%,
4=11-25%, 5=26-50%, 6=51-75%, and 7=76-100%.

Maize hybrid screening trial

The screening of hybrids was conducted under field trials in both rainy and dry seasons in

2017. Maize hybrid of 20-precommercial and 3-commercial varieties belonging to NCSRC, Kasetsart

Univercity were screened by natural disease infection at 6 locations in 4 provinces where those fungal
target isolates were typical samples collected. The field set up for all experiments were arranged is
randomized complete block designs with three replications. Individual plots were 5 m long, 3 m wide
and consisted in four rows planted to 25 seeds per row as previously discribed. When plant reached
physiological maturity, all cobs were harvested per variety. Immediately after harvesting, maize ear
rot infections were evaluated on site based on the symptoms and nature of damage. Disease severity
of cob rot in each ear in the sample was assessed using the disease rating of Reid and Hamiton (1996)
as above mentioned. The incidence of infected cobs per farmstead was calculated using the following
formula: Cob rot incidence = 100(x/N) where, x the number of infection cobs with a rating of 2 or
more and N total number of cobs in maize sample.

On-farm visual assessment

A set of 8-maize commercial varieties were evaluated at 4 districts in 4 provinces in rainy
season (2017) under natural infection. The selection of districts was based on maize production levels
and accessibility. Maize ear rot infections were evaluated on site based on the symptoms and nature of
damage. Disease severity of cob rot in each ear in the sample was assessed using the disease rating of
Reid and Hamiton (1996).

ELISA assay for fumonisin production

The grain maize samples of 8 commercial varieties from on-farm visual assessment were used
for fumonisin production by natural isolates infected. Extraction procedures and ELISA kit quantified
for fumonisin production analysis were carried out based on method described by Berardo et al.
(2011) and the manufacturer’s instructions (Romer Labs Singapore Pte. Ltd.). To obtain each sample
solution for fumonisin analysis, 100 mL of methanol/water (70:30, v/v) was added to 20 g ground
kernel sample of field-test-infected ears. The mixture was then shaken vigorously for 3 min on shaker
and the extract was filtered through a Whatman No.1 filter. The samples were tested with the
AgraQuant® Total Fumonisin Assay (Romer Labs Singapore Pte. Ltd.), which detected total
fumonisins (FB1, FB2 and FB3) at concentrations as low as 0.25 ppm. Data of fumonisins content were
averaged across two replicates.

Data analysis

The maize ear rot incidence and severity and mycotoxin concentration were analysed
separately using a Duncan’s Multiple Range Test (DMRT) (P=0.05). Responses from the collection
were analysed by the SX Statistic Program version 8.

Results and Discussion

Occurrence of ear rot and identification

Ear rot of maize was observed at all locations surveyed with 100% disease prevalence. The
highest incidence was 100% in Chuntuk, Pak Chong, Nong Bun Mak, and Phop Phra, where the lowest

25% also at plot sites (Tambon Pak Chong) in Pak Chong district. Over the two years of study, the most
severity of ear rot was 38% at Chuntuk, Pak Chong district followed by 2 locations Phop Phra, and
Nong Bun Mak, of 35 and 28% respectively (Table 1). Data obtained provided the documentation of ear
rot incidence and its geographic distribution among 6 crop districts in Thailand. This also indicates that
fungal disease associated with maize ears is quite high during the growing seasons studied.

Table 1 Distribution of 138 isolate of Fusarium spp., disease incidence, disease severity, and rainfall of each
location in 2016.

Geographic Origin Number of Disease Disease Rainfall (mm)
Chuntuk, Pak Chong, Nakhon Ratchasima isolate incidence severity
116.5
23 100% 38% 105.9
25% 9% 105.6
Pak Chong, Pak Chong, Nakhon Ratchasima 23 100% 28% 217.3
100% 35% 148.9
Nong Bun Mak, Nakhon Ratchasima 23 130.7
27% 8%
Phop Phra, Tark 23
26% 5%
Muak Lek, Saraburi 23

Khok Charoen, Lop Buri 23

One hundred and thirty eight isolates of Fusarium spp. from infected maize grains from 6
districts were identified for their species level. The results showed that cultural and morphological
identification of pathogenic fungus preliminary revealed F.verticillioides was the causal agent of all
isolates obtained from infected samples. F. verticillioides is a saprophyte (not responsible for maize
ear rot) and a parasite of maize; it can be found as a systemic endophyte in a symptomless biotrophic
state or as a hemibiotrophic pathogen depending on environmental conditions (Bacon et al, 2008).
The symptoms were mostly present on the husk/kernels in the form of symptoms as a white to pink or
salmon-colored, cottony mold that occurs on single or multiple kernels scattered or clustered on the
ears. Infected kernels are frequently tan or brown or have white streaks. Regardless the occurrence of
symptoms, the presence of this fungus in maize constitutes an imminent risk due to its ability to
produce fumonisins, mycotoxins with proven on cancer as possible carcinogen to human (Voss et al,
2002).

Pure colonies of F. verticillioides after 4 days on potato dextrose incubation at room
temperature (25๐C +2๐C), are is white, cottony, and tinged with purple. After 7- day incubation, and

conidiation showed microconidia abundant of single-celled and oval formed in false heads on
monophialides. Macroconidia present with sickle- shaped to straight of 3-5 septate.

A pathogenicity test was conducted on varieties WS4452 (susceptible), NS3 (moderate
resistance), and CP888 (resistance). All 6 isolates of F. verticillioides identified as pathogenic and
caused ear rot symptoms with a significantly different disease severity from the control (P ≤ 0.05)
were used as inoculum for pathogenicity assays. On the susceptible crop (WS4452), isolates F14, F23,
and F40 induced the highest disease severity scores of 6.7, 6.0 and 6.3, respectively. The left isolates
showed disease severity scores of 1.6-5.6 (data not shown). All treatments were found significant
difference (P ≤ 0.05) from the control. After 21 days of artificial inoculation, no symptom was
detected on control ears of nontreated controls. In contrast, the ears inoculated with all isolates of F.
verticillioides showed typical symptoms of ear rot disease. All tested isolates caused different degrees
of ear rot symptoms on cobs. These isolates were completely succeeded for proof of pathogenicity
procedures (Zainudin et al. (2016).This result confirms the identification of causal fungus at first as F.
verticillioides and the inoculation method used to produce ear rot symptoms in this study is also
effective.

Responses to F. verticillioides infection in inbred lines

Evaluation under artificial inoculation, the silk channel inoculation method conducted at NCRSC
which the 60 Fusarium-inoculated inbred lines were determined at maturity during rainy season in
2017. All data reported represent averages across replication. Fusarium ear rot severity for all 60 inbred
lines ranged from 2 (1 to 3%) to 7 (i.e., 76 to 100% ear rot symptoms), and almost all of the entries had

severity levels more than 10%. Only one out of 60 inbred lines (Ki30) showed low disease severity
(rating 2.6). The results showed that most of Ki1to Ki60 lines had high disease severity, were ranked as
susceptible and highly susceptible to Fusarium ear rot. Because the inbred were tested in rainy season
then, high favorable environment contributed to their disease severity induction. According Sutton
(1982) reported environmental factors such as rainfall and temperature were affected the severity of
Fusarium spp. infection. The difference in disease severity was however attributed to differences in
environmental conditions affecting disease development and infection. Fusarium ear rot is most severe
under hot, dry weather conditions that occur after flowering (Gxasheka et al., 2015). Moreover, Sweets
and Wright (2008) point out that most of the fungi are more prevalent when the rainfall is above
normal during silking to harvest.

Another factors that may have contributed to differences in the reaction of inbred lines to ear rot
is pathogen diversity. The most virulence isolate F14 used as inoculum with an effective silk channel
inoculation method with minor modified in this study showed adequate disease assessment critical for
evaluation of maize resistance for ear rot. Resistant inbred line Ki30 was generated enough disease
pressure and overcoming specific source of resistance. Validations of Fusarium ear rot phenotypes of
Ki30 can therefore, facilitate development of breeding program to further improve F.verticilloides
resistance in maize.

Maize hybrid evaluation experiments

Maize plants examined for resistant levels of 20-precommercial (hybrid) and 3-commercial
cultivars by natural infection were conducted under field trials at 6 locations in 4 provinces where
outbreak of Fusarium ear rot has previously been observed. Rainy season experiments resulted in higher
mold severity scores than dry season of the years (data not show) according to natural endermics of ear
rot. Three out of 6 locations at Chuntuk, Park Chong, Nong Bun Mark, and Phop-Phra districts showed
100% ear rot incidence. Most precommercial hybrids from NCSRC showed higher severity than
commercial varieties. However, the effect of two-precommercial varieties, KSX5720 and KSX5911 on
mold severity was no significant difference when compared with those commercial varieties. These two
precommercial varieties possibly revealed moderately resistance tendency present a less severity level
under extreme disease pressure of 100% ear rot incidence in three locations (Chuntuk, Pak Chong,
Nakhon Ratchasima). The commercial hybrids also showed moderately resistance similar to that
occurring in KSX5720 and KSX5911 with 3.6 and 4.0 respectively. The majority of the hybrids was
ranked as either susceptible or highly susceptibl, the other varieties tested appeared to be high severity
of ear rot infection (Table 2). A trend of declining disease incidence and severity found in these
moderate resistance varieties indicated the ear rot resistance expressed in their genotypes that the
development of disease symptoms began to reduce the extent of F.verticilloides colonization under high
disease pressure of infection by different fungal isolates naturally epidemic in the location tested sites
(data not shown).

Table 2 Disease incidence and disease severity of Fusarium ear rot on pre-commercial field maize
under natural infection in 6 locations determined

Location Chuntuk, Pak Chong, Nong Bun Mak Phop Phra Muak Lek Khok Charoen
Pak Chong Pak Chong Nakhon Tark
Maize cultivar3/ Saraburi Lop Buri
1. KSX5402 Nakhon Nakhon Ratchasima DI DS
Ratchasima Ratchasima 100 4.6 DI DS DI DS
DI1/ DS2/ DI DS DI DS 22.3 3.0 30.0 2.0
20.0 2.6 100 5.3
100 6.3

2. KSX5603 100 5.0 30.3 4.3 100 6.0 100 5.0 40.7 3.6 20.0 2.0

3. KSX5614 100 4.3 24.3 3.0 100 4.0 100 3.3 30.3 3.3 31.0 2.0

4. KSX5720 100 3.6 30.0 3.0 100 3.6 100 3.3 20.0 2.0 20.0 2.0

5. KSX5805 100 5.6 44.3 4.3 100 6.0 100 6.0 30.6 3.3 21.0 2.0

6. KSX5819 100 4.6 20.0 2.6 100 5.0 100 4.3 23.3 2.3 35.0 2.3

7. KSX5901 100 6.0 28.0 3.6 100 5.0 100 5.3 65.0 4.6 21.6 2.0

8. KSX5902 100 5.0 33.0 4.0 100 6.0 100 3.3 25.0 2.0 20.0 2.0

9. KSX5903 100 4.6 16.0 2.6 100 4.6 100 4.6 34.3 3.3 39.3 2.3

10. KSX5904 100 5.0 N N 100 4.3 100 5.3 26.6 2.6 21 2.0

11. KSX5906 100 5.0 12.3 2.6 100 5.6 100 5.0 31.6 3.0 26.6 2.0

12. KSX5908 100 4.3 17.6 3.0 100 5.3 100 4.3 23.3 2.3 31.6 2.3

13. KSX5909 100 4.3 20.0 2.6 100 4.6 100 3.0 33.3 3.3 41.0 3.6

14. KSX5911 100 4.0 23.6 3.0 100 4.0 100 4.0 13.0 1.6 20.0 1.6

15. KSX5912 100 4.6 20.0 3.0 100 5.0 100 3.6 18.6 2.0 19.7 1.6

16. KSX5919 100 4.6 18.0 2.3 100 4.6 100 4.3 25.0 3.0 20.0 2.0

17. KSX5924 100 4.6 30.0 3.3 100 5.0 100 4.3 31.0 3.0 50.0 3.6

18. KSX5927 100 5.3 43.3 4.0 100 6.0 100 5.0 28.3 3.3 20.3 3.0
19. KSX5934 100 5.3 22.6 3.3 100 6.0 100 5.3 24.3 3.0 23.3 3.0

20. KSX5937 100 5.0 53.3 4.0 100 4.6 100 4.0 18.3 3.0 24.7 2.3

21. NS3 100 4.0 15.6 2.6 100 2.6 94.3 3.3 13.3 2.6 20.3 2.0

22. CP888 100 3.6 10.0 2.0 100 3.6 91.7 2.6 22.3 2.6 22.7 1.6

23. SW4452 100 5.0 10.0 2.6 100 4.3 96.7 3.6 20.0 3.3 26.7 2.3

% CV 0.00 10.40 12.23 13.78 0.00 10.59 1.34 12.29 15.42 15.86 12.07 17.18

1/ Mean of disease incidence (%).
2/ Mean of disease severity score (1=0% infection, 2=1-3%, 3=4-10%, 4=11-25%, 5=26-50%,

6=51-75%, and 7=76-100%).
3/ No 1 to 20 are precommercial and 21-23 = commercial varieties.

On-farm visual assessment and fumonisin analysis

The present work addressed the effects of Fusarium ear rot on commercial hybrids of field-
grown maize and fumonisin content of affected grains at harvest from five consecutive growing
locations in four provinces. The average of Fusarium ear rot severity of 8 commercial hybrids
analyzed at maturity after harvested was shown in Table 3. The affected husks on grains were
randomly selected from the sites that showed low disease severity from those five locations. Of 8
commercial hybrids tested, SW4452 was shown as high level of disease severity (ranked=5) at

Wang Thong districts. Investigation of fomonisin accumulation in infected grains using ELISA

analysis revealed that all of commercial maizes tested had low level of fumonisin concentration. In
some location samples however, fumonisin was detected with over the 5 ppm concentration levels. A
maximum of 20.61 ppm of fumonisin was detected in PAC139 grains and mean of disease severity
ranked with 4 level at Wang Thong districts. However, varieties PAC559, SW4452, and DK6818 had
high ranked of disease severity (4 and 5 levels) but their low fumonisin accumulation was observed.
These results seem to indicate that no correlation between Fusarium ear rot severity (visualization)

and concentration of fumonisin accumulated in grains (ELISA). Similar results were obtained by
Clement et al. (2004) when testing the correlation between fumonisin production and the severity of
ear rot symptoms. These results suggest that fumonisin production by F.verticilloides under natural
(uncontrolled) condition is not a consequence of the severity of ear rot symptoms in maize grains.
Thus, a toxigenic potential present in this parasitic fungus from Thailand is complex and has a chance
to vary in the range of fumonisin contaminated in food and feeds. Further study should further
conduct whether (i) the virulence effect of F.verticilloides isolates in fumonisin production, (ii) the
tendency quantified in number of isolate populations compared to the visible symptoms and
mycotoxin accumulation, (iii) fumonisin production under both in vitro and in vivo culture conditions,
and (iiii) fumonisin accumulation in grains of resistance and susceptible maize plants, were
responsible for development of ear rot severity and fumomisin production. Data obtained could
address the stability of reduced mycotoxin accumulation in maize grains.

We observed that high humidity, number of precipitation, and temperature likely contributed
to development of ear rot epidemics in different locations resulting variation in the rank among
moderately susceptible and susceptible phenotypes. Current study revealed that the severity of ear rot
epicdemics in rainy season (August-October) was greater than dry season in December-February. This
is in agreement with those obtained by Czembor et al. (2015) that the temperature and rainfall are the
main factors affecting the development of Fusarium species causing important diseases of maize and
other small grain cereals.

This study is the first attempt to identify a causal fungus of ear rot epidemics in Thailand and
evaluate a subset of maize phenotypes classified as moderate resistance and susceptible to
F.verticilloides infection. The capacity of this parasitic fungus to induce ear rot symptoms under field
grown maize in correlation to its fumonisin production was also described. Although more works are
needed to be further elucidated, results of the current study are validated to breeding program and
growers due to a few sources of ear rot resistance have been identified to date.

Table 3 Fusarium ear rot severity and fumonisin concentration of 8 commercial maize hybrids
evaluated after harvest in 2017

District Si Satchanalai Pak Chong Tak Fa1 Tak Fa2 Wang Thong

Commercial Fusarium Fumonicin Fusarium Fumonicin Fusariu Fumonicin Fusarium FumonicinFusarium Fumonicin
maize hybrid ear rot (ppm) ear rot (ppm) m ear rot (ppm) ear rot (ppm) ear rot (ppm)

1. PAC139 21/ 11.83 2 0.17 3 0.93 2 4.47 4 20.61

2. PAC129 2 1.42 2 4.07 3 1.41 2 2.05 2 0.56

3. CP888 2 1.03 2 0.21 2 0.06 2 5.91 2 3.37

4. DK9898 2 0.50 3 1.0 3 0.85 3 7.58 3 8.74

5. NS3 2 3.00 2 1.99 2 0.25 2 1.14 3 0.58

6. PAC559 2 8.25 3 2.29 3 1.89 3 5.11 4 5.70

7. SW4452 2 3.64 2 1.65 3 1.47 4 1.48 5 1.48

8. DK6818 2 0.71 3 2.25 3 5.73 3 2.63 4 1.69

1/
Mean of disease severity score (1=0% infection, 2=1-3%, 3=4-10%, 4=11-25%, 5=26-50%,

6=51-75%, and 7=76-100%).

Acknowledgments

This research was undertaken with the financial assistance of Kasetsart University Research
and Development Institute and National Corn and Sorghum Research Center.

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