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Applied and Environmental Microbiology, May 2009, p. 3289-3295, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02287-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic Diversity and Fitness of Fusarium graminearum Populations from Rice in Korea

Jungkwan Lee,¹ In-Young Chang,¹ Hun Kim,¹ Sung-Hwan Yun,² John F. Leslie,³ and Yin-Won Lee^1*

Department of Agricultural Biotechnology and Centers for Fungal Pathogenesis and for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea,¹ Department of Medical Biotechnology, Soonchunhyung University, Asan 336-745, Republic of Korea,² Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506-5502³

Received 6 October 2008/ Accepted 11 March 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.

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Fusarium graminearum (teleomorph: Gibberella zeae) causes head blight of small grains, including rice, wheat, and barley (23). The fungus was first reported on rice in Italy by Cattaneo (4) as Botryosphaeria saubinetii Niessland. This rice disease has since been recorded in other countries, including Brazil, China, India, Japan, Nepal, and Uganda (11, 31). The disease usually does not cause heavy damage, but under conditions that favor disease development, e.g., high humidity, it may be severe. Chung et al. (7) found that an isolate from wheat could infect rice and other plants and also could cause a postemergence blight in rice. Wheat isolates of F. graminearum can cause significant disease on rice, but under greenhouse conditions no trichothecenes were detected in the infected rice florets (14). In addition, Nepalese rice contained no detectable contamination with trichothecenes even though F. graminearum occurs in Nepal (11).

The fungus can produce the 8-ketotrichothecene mycotoxins nivalenol (NIV) and deoxynivalenol (DON). Most of the biosynthetic genes for the synthesis of 8-ketotrichothecenes are tightly linked in the TRI gene cluster (9). TRI7 and TRI13 are required for acetylation and oxygenation of the oxygen at C-4 to produce NIV and 4-acetyl nivalenol (4-ANIV), respectively, from DON. PCR-based methods to identify polymorphisms in both genes were developed as simple, reliable diagnostic tools for differentiating strains with DON and NIV chemotypes (20, 21). There are regional differences in the distribution of the two chemotypes. Maize and wheat in North America and Europe commonly are contaminated with DON (9), while strains with NIV chemotypes are commonly recovered from cereal crops in Asia (15, 17). In the Republic of Korea, strains with the DON chemotype often cause maize ear rot, while strains with the NIV chemotype commonly are recovered from barley (17, 35). A severe epidemic of Fusarium head blight on wheat and barley occurred in 1963 in southern Korea (5, 6). Humans and farm animals consuming moldy cereals exhibited typical signs of trichothecene intoxication involving vomiting, dizziness, nausea, abnormal pain, and diarrhea (9). The natural occurrence of NIV and DON has been reported in barley and maize in Korea (17, 35, 41), but there have been few surveys of Fusarium mycotoxins in Korean rice.

O'Donnell et al. (30) divided F. graminearum into seven phylogenetic lineages based on the genealogical concordance of six genes. The phylogenetic separation has been used to raise these seven and four additional lineages to species status (36). The geographic location often influences the lineage present, e.g., lineage 7 is the most common in the United States, and lineage 6 dominates in China. Lineage and trichothecene chemotype are not correlated (45), and the lineages are morphologically cryptic. Members of all lineages are cross-fertile with strains belonging to lineage 7 and in some cases with strains of other lineages (1, 2, 19, 25), a pattern that suggests that the members of all of the lineages belong to a single biological species.

Studies of F. graminearum populations have been made in different geographic regions, e.g., China (12), Europe (42), the United States (48, 49), and Argentina (34). Populations of F. graminearum have high levels of genotypic diversity, which suggests that recombination occurs regularly in F. graminearum populations. Most studies have focused on populations from wheat, barley, and corn, and there is little information on F. graminearum populations from rice.

Severe epidemics of Fusarium head blight of rice occurred in August 2001 after heavy rainfall during the rice flowering period in southern Korea. Lesions on or discoloration of the glumes were common, with infected grains first appearing to be white and later yellow, salmon, or carmine. Sometimes the entire seed was colonized. Infected grains were lightweight, shrunken, and brittle. Our objectives in the present study were (i) to determine the frequency at which F. graminearum occurs in plants with rice head blight; (ii) to determine the number and relative frequency of the F. graminearum lineages present; and (iii) to evaluate the strains for their sexual fertility, genetic relatedness, virulence, and toxin-producing abilities. Our working hypotheses were that sexually fertile strains from lineage 6 would dominate in the population and that these strains would be the most aggressive toward rice. We expected most of the lineage 6 strains to produce NIV and for there to be a high level of genetic variation, as assessed by neutral (amplified fragment length polymorphism [AFLP]) markers. We evaluate here F. graminearum population diversity in Korea and provide new information on the pathogenic capabilities of strains belonging to several of the known lineages of this very widespread fungal species.

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Rice samples and strains.
Rice seeds, including the husk, with head blight symptoms (500 g per sample) were collected from 48 rice fields in four southern Korean provinces (Gyeongnam, Gyeongbuk, Jeonnam, and Jeonbuk) during August and September 2001. Seeds of each sample were collected from 200 different rice plants. To compare the lineage composition, toxin production, and fertility of the isolates geographically, five rice samples also were collected in Kangwon province in eastern Korea during August and September 2002, where maize is a major part of the farming system. All of the rice samples were dried and stored at 4°C until the fungal strains were isolated. Fourteen strains of F. graminearum from rice in Mississippi were provided by H. Abbas. Fourteen F. graminearum strains were used as reference strains for lineages 1 to 7 (NRRL strain numbers 11796 and 11797 for lineage 1, 11794 and 11795 for lineage 2, 11791 and 11792 for lineage 3, 11789 and 11790 for lineage 4, 11787 and 11788 for lineage 5, 11785 and 11786 for lineage 7, and 11157 and 11784 for lineage 7) in the AFLP analyses.

Isolation of fungi.
One hundred seeds with head blight symptoms from each sample were soaked in 1% sodium hypochlorite for 2 min, rinsed in sterile water for 2 min, placed on the surface of a plate of peptone-PCNB medium (23), and then incubated at 25°C for 4 to 7 days. Fusarium isolates were transferred to potato dextrose agar and carnation leaf agar (CLA) and then incubated under a fluorescent cool-white light (5,000 lx) at 25°C (23). All cultures were purified by subculturing single conidia, and the resulting cultures were stored as spore suspensions in 20% glycerol at –80°C.

Sexual fertility tests.
CLA and carrot agar were used to test the sexual fertility of each isolate (23). Each isolate was grown on CLA for 20 days at 25°C under a fluorescent cool-white light (5,000 lx) with a 12-h photoperiod. Each isolate also was grown on carrot agar under the same conditions for 7 days, at which time the mycelia were knocked down with a glass rod after the addition of 1 ml of a 2.5% Tween 60 solution. The carrot agar plates were returned to the incubator and incubated for two additional weeks, after which the production of perithecia and ascospores were evaluated microscopically. The test was repeated at three different times with three replicates on both CLA and carrot agar for each isolate.

DNA isolation and AFLP.
DNA was isolated with a CTAB (cetyltrimethylammonium bromide) procedure (23). We estimated final DNA concentrations (in Tris-EDTA buffer) by comparison of DNA fluorescence of diluted aliquots of each DNA sample against that of HindIII-digested bacteriophage DNA with an IS-1000 version 2.0 digital imaging system (Alpha Innotech, San Leandro, CA). Samples and sample dilutions were run in 1% agarose gels containing TAE (40 mM Tris-acetate, 1 mM EDTA [pH 8.0]) and 0.5 µg of ethidium bromide/ml. The concentration of each DNA sample was adjusted to 20 µg/ml for use in the AFLP analyses.

AFLPs were generated with the protocol of Vos et al. (44) as modified by Leslie and Summerell (23). AFLP primers were synthesized by Integrated DNA Technologies (Coralville, IA). The EcoRI primers in the final specific amplification reactions were 5' end labeled with [-³³P]ATP (NEN Life Sciences, Boston, MA). Dried gels were exposed to X-ray film (Classic Blue Sensitive; Molecular Technologies, St. Louis, MO) for 2 to 5 days at room temperature to identify DNA bands. We manually scored the presence or absence of polymorphic AFLP bands representing DNA fragments between 200 to 800 bp (bp) in length and recorded the allele data (presence or absence) in a binary format. Bands of the same size were presumed to be homologous. We estimated molecular weights of AFLP fragments by comparison to the Low Mass Ladder (Life Technologies, Bethesda, MD) DNA standard that was 5' end labeled with [-³³P]ATP.

Population genetic analyses.
We identified AFLP haplotypes (putative clones) within populations by analyzing the binary data with the unweighted pair grouping by mathematical average (UPGMA) subroutine of PAUP* 4.0, beta 10 (39). Bootstrap analyses (1,000 iterations) were conducted on the resulting UPGMA tree to assess the support for any resulting subgroups. We estimated the allele frequencies at polymorphic loci (28), the Nm values (effective migration rate [26[), and the genetic identity among populations (29) using the shareware program POPGENE version 1.32 (47). We also estimated genotype diversity () for each population as described by Milgroom (27) and normalized the index for each population by dividing each estimated by the number of genotypes identified from that population.

PCR assay of trichothecenes.
DON and NIV chemotypes were determined by PCR with the Tri7 and Tri13 alleles in the trichothecene biosynthetic gene cluster as previously described (20, 21). Tri7-specific primers were GzTri7/p1 (5'-GGCTTTACGACTCCTCAACAATGG-3') and GzTri7/p2 [5'-G(A/G)CGG(C/T)AAAGAAAACCAATCAAC-3']. Tri13-specific primers were GzTri13/p1 [5'-AATACTA(A/C)AAG(C/T)CTAG(G/T)ACGACGC-3'] and GzTri13/p2 [5'-GTG(A/G)T(A/G)TCCCAGGATCTGCGTGTC-3']. Oligonucleotides were synthesized by Bioneer Corp. (Chungwon, Republic of Korea), dissolved at 100 µM in sterile water, and stored at –20°C. For PCRs, 100 ng of genomic DNA was used as a template in a 50-µl reaction containing 2 mM MgCl₂, 1x rTaq PCR buffer (Takara Biomedicals, Shiga, Japan), deoxynucleoside triphosphates at 0.2 mM each, primers at 2 µM, and 1.25 U rTaq (Takara Biomedicals). PCRs were performed in a thermal cycler (MJ Research, Waltham, MA) with an initial denaturation step at 95°C for 2 min; 30 cycles of 94°C (1 min), 55°C (1 min), and 72°C (3 min); and a final extension step at 72°C for 10 min. F. graminearum lineage 7 strain H-11, a DON producer isolated from maize in Korea, and F. graminearum lineage 6 strain 88-1, an NIV producer isolated from barley in Korea, were used as standards for the two lineages, respectively (21).

Trichothecene analysis of fungal cultures.
Each isolate was screened for trichothecene production on rice and toxins were extracted as previously described (40). In brief, the toxins were analyzed with a Shimadzu QP-5000 gas chromatograph-mass spectrometer in full-scan mode. The analytical conditions were as follows: column, DB-5 fused silica column (0.25 mm [inside diameter] by 30 m, 0.25-µm film [J&W Scientific, Folsom, CA]; column temperature, 120°C for 5 min and then increased to 270°C at 5°C/min; injector temperature, 280°C; ion source temperature, 200°C; interface temperature, 250°C; and ionizing voltage, 70 eV.

Pathogenicity test of isolates on rice and maize.
Six isolates (lineage 6, NIV producers) from four populations of the southern provinces, three isolates (lineage 3, DON producers) from maize in eastern Korea, and three isolates (lineage 7, DON producers) from rice in Mississippi were arbitrarily selected for pathogenicity tests on rice and maize in the greenhouse. Conidia were harvested from cultures grown on carrot agar (23) for 2 weeks at 25°C and then suspended in sterile water at a concentration of 10⁵ conidia per ml. For the inoculation of rice heads, 1 ml of the conidial suspension was sprayed on the rice head (cv. Nakdong) with an aerosol sprayer at early anthesis. Plants were placed in a growth chamber for 2 days and grown under a cycle of 14 h in light at 28°C and 10 h in dark at 25°C with 100% relative humidity and then transferred to a greenhouse. Maize (cv. Okcheon) was inoculated with toothpicks soaked in sterile water for 1 h, washed twice with sterile water, and autoclaved for 20 min at 121°C. Sporodochia on carrot agar were scratched with the toothpicks, and the contaminated toothpicks used to inoculate maize plants by piercing the ear's husk 4 to 5 days after silking. The toothpicks remained in the maize ears until the end of the growing season. Ten rice heads and ten maize ears were inoculated with each strain. The experiment was performed twice. Disease severity ratings were based on the percentage of visibly damaged seeds on a rice head or a maize ear: 1, 0 to 20%; 2, 21 to 40%; 3, 41 to 60%; 4, 61 to 80%; and 5, 81 to 100%.

Recovery of F. graminearum from inoculated rice.
Three isolates, one each representing lineages 3, 6, and 7, which had similar levels of aggressiveness to rice and maize, were arbitrarily selected for the adaptation test to rice: RBS12 (lineage 6, NIV producer) from rice in southern Korea, USA17 (lineage 7, DON producer) from rice in the United States, and RMS8 (lineage 3, DON producer) from maize in eastern Korea. Conidia of each isolate were suspended in sterile water at a concentration of 3 x 10⁵ conidia per ml. The three conidial suspensions were combined, and 1 ml of the mixed conidial suspension was used to inoculate rice heads (cv. Nakdong) as described above. Each rice plant was planted in a pot, and a total of 20 plants were inoculated. The plants were placed in a growth chamber for 2 days and then transferred to nursery plots in 2003. Half of the rice heads were harvested at the end of the growing season (November), and the remainder was collected the following spring (March) after overwintering in the nursery plots. Fungal isolates were purified from the seeds as described above, and the lineage of each isolate determined by PCR assay of the alleles present at the Tri7 and Tri13 loci.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Occurrence of Fusarium species in rice seeds.
F. graminearum was recovered from all 48 samples from southern Korea and all five samples from eastern Korea. Fusarium fujikuroi was recovered from 4 of 48 samples from southern Korea but not from any of the samples from eastern Korea. Other Fusarium species also were present at frequencies of <5%, but these strains were not identified to the species level. F. graminearum was found in 2 to 91% of the seeds per sample, with a mean of 43%. A total of 2,064 F. graminearum isolates were detected, and 5 or 6 isolates (total of 249 isolates) were chosen from each sample from southern Korea for further study; 107 isolates of F. graminearum were recovered from rice in eastern Korea.

Fertility.
A total of 71% of the isolates tested from southern Korea produced perithecia homothallically on either CLA or carrot agar, while the remaining 29% never produced perithecia even when the incubation was continued for four additional weeks (Table 1) . The percentage of fertile homothallic strains (69%) from the eastern Korean samples was similar to that for the southern Korean samples (Table 1). The number of perithecia produced by the fertile isolates was low, with usually only a few perithecia produced on the carnation leaves or the carrot agar. Ten of fourteen isolates from rice in Mississippi produced abundant perithecia, while the other four isolates did not produce any perithecia (Table 1).

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TABLE 1. PCR assay, chemical analysis, and fertility of F. graminearum isolates from rice in Korea and the United States

Population structure and genetic diversity.
Three primer pair combinations (EAA/MAT, ECC/MCG, and ETG/MTT) resulted in 227 AFLP bands. In a UPGMA analysis of the AFLP banding patterns, all 249 isolates from southern Korea and 84 of 107 isolates from rice in eastern Korea clustered with the standard isolates of lineage 6. The remaining 23 isolates from eastern Korea and all 14 isolates from rice in Mississippi clustered with the standard isolates of lineage 7.

Of the 227 AFLP bands, 82 (36%) were polymorphic, and the allele frequencies were very similar across the four southern Korean populations and the eastern Korean population, excluding the 23 lineage 7 isolates. Among the 333 lineage 6 isolates from the five populations, there were 270 multilocus haplotypes, 225 of which were represented by a single strain and 45 of which were detected more than once. Of the 45 multiply represented haplotypes, 40 were found at only one location. Five of the multiply represented haplotypes were found in two different provinces. Nei's unbiased measures of genetic identity among the five populations were high, ranging from 0.972 to 0.998 (Table 2), indicating little genetic differentiation. The effective migration rate (Nm) ranged from 2 to 25 (Table 2). Similar results were obtained when we analyzed just a subset of 45 loci for which the frequency of both alleles (presence or absence) was >5% (Table 2). The number of private alleles, i.e., alleles present in one population but not in any of the other populations, ranged from 0 to 11 among the five Korean populations. The frequencies of the private alleles were all <5% except for six alleles in the eastern Korean population (Table 3).

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TABLE 2. Nei's unbiased measures of genetic identity for five populations of F. graminearum from Korea

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TABLE 3. Estimates of genotypic diversity and the numbers of pairs of significant linkage disequilibria in five Korean populations of F. graminearum based on AFLP loci

Genotypic diversity also varied by population. The highest genotypic diversity (58%) was in the Jeonnam population and the lowest (30%) was in the Kangwon population (Table 3). The number of polymorphic loci in an individual population ranged from 32 to 56. The percentage of locus pairs in significant linkage disequilibria (² test, P < 0.05) in the five populations ranged from 14% (173 of 1,275 pairs) in the Gyeongnam population to 22% (154 of 703 pairs) in the Jeonbuk population (Table 3). At the P < 0.01 significance level, the percentage ranged from 7% (35 of 496 pairs) in the Gyeongbuk population to 17% (116 of 703 pairs) in the Jeonbuk population. If the 45 loci with both alleles present at >5% only were used for this analysis, then the percentage of loci in disequilibrium (² test, P < 0.05) ranged from 16% (109 of 666 pairs) in the Gyeongnam population to 32% (161 of 496 pairs) in the Kangwon population (Table 3). At the P < 0.01 significance level, the percentage ranged from 9% (63 of 666) in the Gyeongnam population to 24% (118 of 496) in the Kangwon population.

PCR assay and chemical analysis for trichothecene production.
Tri7- and Tri13-specific primer pairs were used to differentiate strains with the NIV and DON genotypes. Of the 249 isolates from rice in southern Korean provinces, all but 8 had the NIV genotype, while 13 of 14 isolates from the United States had the DON genotype (Table 1). Chemical analysis was consistent with the PCR diagnoses in all but a few cases. All 241 Korean isolates with the NIV genotype also produced NIV, and all except 6 isolates also produced 4-ANIV. Of the eight isolates with the DON genotype, seven produced both DON and 3-acetyl deoxynivalenol (3-ADON), and one produced no trichothecenes. Of the 107 isolates from rice in eastern Korea, the 84 lineage 6 isolates produced NIV and the 23 lineage 7 isolates all produced DON (Table 2). Eighty-three isolates of the eighty-four NIV producers also produced 4-ANIV. Of the 23 DON producers, 5 isolates produced 3-ADON and 18 isolates produced 15-ADON. Among the 14 isolates from the United States, the strain with the NIV genotype produced NIV and 4-ANIV, 12 strains had the DON genotype and produced DON, and 1 strain produced no trichothecenes. Of the 12 of DON producers, 6 isolates produced DON and 3-ADON, 3 isolates produced DON and 15-ADON, and 3 isolates produced only DON.

Pathogenicity of isolates on rice and maize.
Head blight symptoms on rice heads infected by F. graminearum appeared 1 week after inoculation. Symptoms began at points as water-soaked brown spots, with bleaching becoming apparent within a few days. Disease severity was counted 4 weeks after inoculation. All isolates caused head blight with severity ratings of 3.2 to 3.6, where there were no significant differences (P < 0.05) based on a Tukey test as implemented in SPSS 12.0 software (SPSS, Inc., Chicago, IL). The maize cultivar Okcheon was extremely susceptible to all of the isolates tested. Whole seeds of each ear were completely covered by mycelia. All isolates caused severe ear rot, with a severity rating of 5 at 60 days after inoculation.

Recovery of F. graminearum from infected rice seeds.
From the pathogenicity test, we selected three isolates, PMS8 (lineage 3, DON producer), RBS12 (lineage 6, NIV producer), and USA17 (lineage 7, DON producer), which had similar levels of disease severity on both rice and maize. A mixed fungal inoculum, composed of equal numbers of conidia from the three isolates, was used to inoculate rice heads in the nursery plot during flowering in August. Rice grain with blight symptoms was harvested from the inoculated plants in November, and fungi were recovered from 10 to 15 seeds per plant. We did not try to recover F. graminearum isolates from the mock-inoculated plants, which had no disease symptoms on their heads. A total of 291 F. graminearum isolates were recovered from the harvested seeds. The strains differ in toxin genotype: PMS8 was Tri7^NIV Tri13^DON, RBS12 was Tri7^NIV Tri13^NIV, and USA17 was Tri7^DON Tri13^DON. If toxin alleles are used to indicate lineage, then 3% of the recovered isolates were lineage 3, 42% were lineage 6, and 55% were lineage 7. After overwintering, i.e., in March, the remaining rice seed was harvested and F. graminearum strains recovered. Of the 217 isolates of F. graminearum recovered at this time, 3% were lineage 3, 45% were lineage 6, and 52% were lineage 7 based on the Tri7 and Tri13 alleles present.

Abundant perithecia were produced on the lower portions of the rice plants after overwintering (Fig. 1). Ten to fifteen perithecia per plant were selected, and single-ascospore isolates of F. graminearum were isolated from the perithecia (one spore per perithecium) formed on rice straw. Of these 247 isolates, 87% were lineage 6 and 13% were lineage 7, with no lineage 3 ascospores identified based on the Tri7 and Tri13 alleles.

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FIG. 1. Perithecium production on the lower portion of rice stems after overwintering. Rice was inoculated with a conidial mixture of strains from F. graminearum lineages 3, 6, and 7 at flowering. The inoculated plants overwintered in nursery plots. The picture was taken in the spring (March) of the following year.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The symptoms of Fusarium head blight of rice are typical of those caused by this fungus on other cereals, e.g., wheat and barley. The symptoms also resemble the rice grain rot caused by Burkholderia glumae, which is the most important bacterial disease of rice in Japan, the Republic of Korea, and Taiwan (43). The similarity of symptoms and the relative rarity of Fusarium head blight could lead to an underestimation of the importance of Fusarium head blight on rice in Korea. Severe epidemics of head blight of rice have occurred since 2001 in southern Korea. The increase of Fusarium head blight epidemics in rice could result from changes in cultural practices or climate. Earlier planting of rice enables double-cropping with barley but also results in rice flowering during the rainy season, which favors Fusarium head blight development. The causal agent of the head blight epidemics in our study was F. graminearum not B. glumae. All of the rice samples with head blight symptoms were infected with F. graminearum. Thus, this report is the first evaluation of epidemics of rice head blight caused by F. graminearum in Korea.

We observed no difference in aggressiveness toward rice and maize between isolates of lineages 3, 6, and 7, which is consistent with the hypothesis that aggressiveness is not lineage specific. Several studies have suggested that aggressiveness of F. graminearum may be correlated with toxin chemotypes. In seedling assays, Chinese isolates of F. graminearum lineages 6 and 7 also showed similar levels of aggressiveness toward wheat (33), and the aggressiveness was not correlated with lineage, toxin production, or chemotype under greenhouse conditions (14). In another seedling assay (3), however, lineage 6 strains of F. graminearum with the NIV chemotype were significantly more aggressive toward maize than were lineage 6 strains with the DON chemotype. In a recent ear rot experiment (10), lineage 6 isolates with the NIV chemotype tended to be less aggressive to maize than were DON producers. We could not determine whether toxin chemotype within a lineage was an important factor for aggressiveness, because we did not test the aggressiveness of strains with different toxin chemotypes from the same lineage. However, our results suggest that aggressiveness toward the cultivars used in the present study was not related to the lineages. Clearly, aggressiveness is a complex trait that depends on host and environment for its expression, with a great deal of research still needed to resolve the physiological, environmental, and genetic components of this economically important trait.

The lineage composition of F. graminearum populations does appear to be host and location dependent. In eastern Korea, 70% of the F. graminearum isolates from maize belonged to lineage 7 (22), whereas 80% isolates from rice belonged to lineage 6. We expected most isolates from the southern Korean provinces to belong to lineage 6 since little maize is grown in this area and since lineage 6 dominates on barley in these provinces. We recovered only lineage 6 isolates from rice in the southern provinces, which is consistent with the hypothesis that F. graminearum lineage 6 has a host preference for rice.

Lineage distribution also may be correlated with annual temperature. The relative proportion of lineages 6 and 7 in the population could depend on annual temperature rather than host preference. In both China (33) and Japan (37), lineage 6 dominates in warmer regions and lineage 7 dominates in cooler regions. In Korea, the population composition also is consistent with this hypothesis since lineage 6 dominates in warmer southern regions and lineage 7 dominates in cooler eastern Korea, but the confounding of host and temperature make it difficult to draw this conclusion without reservations.

The Korean rice populations of F. graminearum have lower levels of genotypic diversity and significantly more clones than did populations reported from China (12) and the United States (13, 46, 48, 49). The clones could result from asexual reproduction, e.g., via conidia, or from less outcrossing and more homothallic sexual reproduction, which is consistent with the higher levels of linkage disequlibrium observed in the Korean populations. Our linkage disequilibrium calculations, however, need to be interpreted with caution since we do not know the locations of the markers on either the physical or the genetic maps of F. graminearum, or their relative genetic distances from one another.

The genetic identity across the populations from southern and eastern Korea was high. Among the populations from southern Korea, there were relatively few private alleles. The eastern Korea population not only had more private alleles, but 6 of 11 private alleles were present at a level of >5%. These results suggest that significant genetic exchange occurs among the populations from southern Korea. Exchanges between the southern and eastern parts of the country also appear to be occurring but at a much lower level, since the number of private alleles in the eastern Korean population is higher than in any of the populations from southern Korea and Nm values associated with these populations and the one from eastern Korea are all relatively small (Table 2). The level of genetic exchange in all of the Korean populations is lower than what has been inferred elsewhere; however, the greater proportion of clones in the Korean populations may make it more difficult to quantify the amount of genetic exchange that is actually occurring among the Korean populations.

The lineage 6 strain we worked with had a clear selective advantage in rice over the strains from the other lineages. If this strain typifies lineage 6, then this advantage could be an important evolutionary adaptation by this group of F. graminearum strains. This selective advantage could occur during any part of the life cycle and need not to be directly related to pathogenicity. The selective advantage could be related to increased sexual fertility on rice because 87% of the perithecia produced on the rice straw in the field were from lineage 6, and only 13% were from lineage 7. This result was unexpected since the in vitro production of perithecia by lineage 6 strains on carrot agar and CLA is only 10% that produced by the lineage 7 strains.

Ascospore production may be an important factor in Fusarium head blight of rice, as has been proposed for the wheat/F. graminearum interaction (32, 38). This apparent difference may be due to differences between strains of lineage 6 and lineage 7, in which case it should be segregating in the mapping population of Jurgenson et al. (16) and could be readily localized on the physical map of F. graminearum (18) as either a single gene or as a series of quantitative trait loci (8). The difference also could be due to differences in host composition (rice versus maize or wheat) or to environmental conditions, e.g., temperature, humidity, and light, that occur when perithecia are initiated. Identification of the physiological and/or genetic factors responsible for this differential fertility could be used to develop host material less suitable for perithecium formation than are those currently available. A reduction in perithecium production could reduce the number of ascospores available as inoculum during the window in which the hosts are susceptible to infection.

We could not determine whether outcrossing occurred in the field since we scored only a single character and then used that character to identify the lineage to which the perithecia belong. F. graminearum is known to outcross under laboratory conditions, and crosses between isolates of different lineages also can be made (1, 16, 19) under these conditions. At present, the evidence for outcrossing under field conditions is indirect, since the populations evaluated all contain numerous strains that differ in genotype. The moderate levels of linkage disequilibrium observed are easiest to explain if outcrossing occurs regularly, but outcrossing is not as common in this population as it is in other Fusarium spp. and F. graminearum populations (24).

Our results are consistent with the hypothesis that F. graminearum populations from rice are composed primarily of isolates of lineage 6. The selective advantage for lineage 6 isolate may not be due to superior ability in pathogenic ability but rather to an ecological ability that need not be directly related to the mechanism of pathogenicity. The basis for rice straw's selective effect for lineage 6 needs to be determined, and the genes responsible for this trait need to be identified at the molecular level. Such genes are potentially important in many Fusarium species and could lead to the elucidation of new host/pathogen interaction models and the development of novel control mechanisms.

 
This study was supported by grant CG1411 from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Korean Ministry of Education, Science, and Technology; by a Korea Science and Engineering Foundation grant from the government of the Republic of Korea (R11-2008-062-01001-0); by the Kansas Agricultural Experiment Station; and by the U.S. Wheat and Barley Scab Initiative.

We thank Hamed Abbas of the Crop Genetics and Production Research Unit, USDA-ARS, Stoneville, MS, for providing us with F. graminearum strains from rice in Mississippi.

This is manuscript 09-102-J from the Kansas Agricultural Experiment Station, Manhattan.

 
* Corresponding author. Mailing address: Department of Agricultural Biotechnology and Centers for Fungal Pathogenesis and for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea. Phone: (82) 2-880-4671. Fax: (82) 2-873-2317. E-mail: lee2443{at}snu.ac.kr

Published ahead of print on 20 March 2009.

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Applied and Environmental Microbiology, May 2009, p. 3289-3295, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02287-08
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