Impact of Crop Rotation on Pathotype and Genetic Structure of Phythophthora sojae in Fields

To estimate the impact of crop rotation on the pathotype and genetic structure ofPhythophthora sojae in fields, 372 isolates ofP. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence ofP. sojaeon 13 differential cultivars, and the amplified fragment length polymorphism (AFLP) technique was used to analyze difference in the genetic structure ofP. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes ofP. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested thatP. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.     

Phytophthora sojae Races in Northeast of China and Virulence Evaluation of the Isolates

42 isolates of P. sojae were obtained from diseased soybean plants in 9 counties of Heilongjiang Province and 1 county of Jilin Province in Northeast of China. 11 isolates were classified into 3 races ;the rest that could not be classified due to intermediate reactions were classified into 12 virulence types, suggesting virulence diversity of P. sojae in China. Isolates represented race 1,3 and 8. Race 1 was the most prevalent one.and races 3 and 8 were the first report in China. Our results suggest P. sojae is aboriginal in China.     

Pathotypes and Genetic Diversity of Blumeria graminis f. sp. hordei in the Winter Barley Regions in China

Barley powdery mildew caused by Blumeria graminis f. sp. hordei （Bgh） is one of the most destructive foliar diseases of barley in the winter barley region of China. The evaluation and assessment of the virulence and diversity of Bgh populations help to determine effective sources of resistance to the pathogen. 515 isolates were collected from seven populations of Bgh on cultivated barley in seven geographically distant locations in 2006. Their virulence was determined by inoculation onto 26 differential host lines. All of the isolates belonged to 58 pathotypes and 13 of which included 81% of these isolates. The most abundant pathotype was pathotype 0047 （18.3%）, the second most abundant was pathotype 0045 （11.8%） and the third most abundant was pathotype 0057 （7.8%）. Most of virulent genes investigated in this study showed similar frequencies in the seven different areas. These indicate that the seven locations may be in a uniform epidemiological region and barley cultivars in these areas may have similar genetic background. Diversities within these populations and distances between these populations measured by KOIND package were different. Correlations were not found between the genetic distance and the geographical distances between different locations. This suggested that long distance spread and local epidemics existed in the major winter barley growing regions in China.     

Sampling Survey and Identification of Races of Soybean Cyst Nematode （Heterodera glycines Ichinohe） in Huang-Huai Valleys

Soybean cyst nematode （SCN Heterodera glycines Ichinohe） is one of the most important nationwide soybean diseases in China. A total of 38 soil specimens or locations in the area was sampled and tested for SCN races during 2001-2003 for the inspection of race distribution in Huang-Huai Valleys. A map of race distribution was constructed according to the data from both the present study and the published reports cited. Three areas, namely, the area of southeast to Jinan in Shangdong Province; the area of northern Henan Province and its border region to south of Hebei Province; and the area of Luohe, Zhoukou of Henan Province and Fuyang of Anhui Province mainly infested with Race 1 were identified. Race 4 was predominant in Shanxi Province, Beijing and the adjacent area of Henan, Shandong, and Anhui provinces, and the delta of Huanghe River in Shandong Province. Race 2 was mainly found in Liaocheng, Dezhou of Shangdong Province and Shijiazhuang of Hebei Province, and Jiaozuo and Huojia of Henan Province. Race 7 was distributed in the west part of Jiaodong Peninsula of Shandong Province and Kaifeng, Huaxian, Wenxian of Henan Province. Race 5 was found and scattered in Hebei and Henan Province. Race 9 was found in Shangqiu of Henan Province, which was reported for the first time in China. It can be seen that Race 1 and Race 4 were the two predominant races in Huang-Huai Valleys, and that research should focus on developing resistant cultivars of these races. There might exist other races in an area with some predominant races. The race substitution in the past decade was not obviously found, therefore, the results should be meaningful to future breeding for resistance to SCN in Huang-Huai Valleys.     

A new pathotype characterization of Daxing and Huangyuan populations of cereal cyst nematode(Heterodera avenae) in China

The cereal cyst nematode(CCN, Heteroder aavenae) causes serious yield loss on cereal crops, especially wheat, worldwide. Daxing population in Beijing City and Huangyuan population in Qinghai Province, China, are two CCN populations. In this study, the CCN pathotypes of Daxing and Huangyuan populations were characterized by tests on 23 standard "International Test Assortment" with the local species Wenmai 19 as the susceptible control. Tested materials were grouped by three nematode populations' virulence on resistant genes(Rha1, Rha2, Rha3, Cre1) and nonresistant genes, varieties and lines. Both Daxing and Huangyuan populations were avirulent to Ortolan(Ha1). Barley cvs. Ortolan, Siri, Morocco, Bajo Aragon 1-1, and Martin 403-2 were all resistant to both populations. Cultivars Herta, Harlan 43 and wheat Iskamish-K-2-light were all susceptible to Huangyuan population, all of them, however, were resistant to Daxing population. The other five oats were all resistant to the two tested CCN populations. Except Iskamisch K-2-light, all the other wheat cultivars(Capa, Loros×Koga, AUS 10894, and Psathias) were susceptible to Daxing population. Because the pathotypes of the two tested CCN populations in Beijing and Qinghai were not identical to any of the 13 pathotypes previously characterized by the test assortment, we classified Daxing and Huangyuan populations as the new pathotypes, named Ha91.     

Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0%were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0%to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated signiifcantly with geographical distance （R2=0.494, P 0.001）. In addition, isolates from Xinjiang appear to form a separate group. Signiifcant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.     

Analysis of the Abnormal Segregation of Pathogenicity in Magnaporthe grisea by Using a Genetic Cross of Oryza and Eleusine Isolates

A genetic cross between Oryza isolate Y93-164a-1 and Eleusine isolate SA98-4 was established, and the pathogenicity of 151 F1 progeny isolates was investigated on both host plants rice and finger millet. Results showed that the segregation of pathogenicity in this genetic cross was abnormal, i.e., most of the progeny isolates were nonpathogenic on both host plants. However, no abnormal segregation was observed when middle repetitive sequence MGR586 and 31 single-copy RFLP markers from all of the chromosomes were genetically analyzed. At the same time, comparison of the chromosomal organization among two pairs of parental isolates did not find any genomic abnormity. These results suggested that the ＂abnormal＂ inheritance of pathogenicity in this cross was most likely due to the reassortment of numerous host species specificity genes but not the biased segregation of the host species specificity genes. The host species specificities in M. grisea were likely to be multigenically controlled, at least in the genetic cross involving rice pathogen and the grasses pathogen other than rice.     

Analysis of Genetic Diversity in Natural Populations of huashanica Keng Using Microsatellite （SSR） Markers

Psathyrostachys huashanica Keng is endemic to China and only distributed in Huashan Mountain in Shaanxi Province, China. In this study, 15 P. huashanica populations consisting of 450 individuals sampled across their main distribution were investigated by using the simple sequence repeats （SSRs） markers. A total of 184 alleles were detected on 24 SSR loci, and the number of alleles on each locus ranged from 2 to15, with an average of 7.667. The total gene diversity （HT= 0.683） and the coefficient of population differentiation （GST = 0.125） showed that P. huashanica had a relatively high level of genetic variation, and the genetic variation was mainly distributed within the populations. The gene flow among the populations of P. huashanica （Nm = 1.750） was much less than that of the common anemophytes （Nm = 5.24）. Correlation analysis demonstrated that the number of alleles as well as genetic diversity of the five populations of Huangpu valley decreased along with the increase of altitudes, but the correlation was not significant. Implications of these results for future P. huashanica collection, evaluation and conservation were discussed.     

Evaluation of the Pathotypes of Xanthomonas oryzae pv. oryzae and Preliminary Analysis of the Resistant Reactions of Main Japonica Rice in the Yunnan Plateau, China

The pathogenicity of 36 isolates of Xanthomonas oryzae pv. oryzae （Xoo）, which were collected from japonica rice varieties in the Yunnan Plateau, China, was evaluated. It was evaluated on 29 rice varieties including a set of seven varieties to identify pathogenicity, i.e., Haonuoyang, TN1, Kogyoku, Zhenzhu＇ai, IR26, Nanjing 33, and Kinmaze, which may be considered as a set of differential varieties for Xoo races from Yunnan japonica rice. The efficiency of the seven varieties was further confirmed. The results showed reversible and specific interactions between isolates and varieties. The isolates were classified into nine pathotypes from pathotyp Ⅰ to Ⅸ according to their pathogenic reactions on the seven rice varieties. The pathotype V was the epidemic, whereas pathogen Ⅶ was the most pathogenic. Most japonica varieties grown in the Yunnan Plateau were susceptible to Xoo. The rice lines IRBB21 （Xa-21）, Zhachanglong （Xa-22,, Xa- 24,）, and IR1545-339 （xa-5）, which were resistant to all the isolates tested, can be used as donors of resistant genes for bacterial blight in japonica rice breeding in the Yunnan Plateau.     

Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

The genetic variation and phylogenetic relationships of Citrus tristeza virus （CTV） isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions （p23, p20, p13, p18, p25, p27, POL, HEL and k17） with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3′ proximal region was relatively conserved, and the 5′ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.     

Development of Chinese Near-isogenic Line of Rice and Their Differentiating Ability of Pathogenic Races of Blast Fungus

The rice( Oryza L. ) variety, Lijiangxintuanheigu(LTH), is a universally susceptible variety. A set of near-isogenic lines (NILs) of rice which has single resistance gene for each line was developed by use of LTH as recurrent parent and Kusabue,Tsuyuake, K1, PiNo. 4, K60 and BL1, which are from Kiyosawa‘s differential varieties(KDV), as donor parents. Differentiating ability of these NILs to Philippine isolates was compared with that of their donor parents, KDV and IRRI‘ s NILs. The results of comparative studies were as follows: (1) Differentiating ability of Chinese NILs to Philippine isolates was much stronger than that of their donor parents and that of KDV. (2) Chinese NILs had similar ability to differentiate races to IRRI‘s NILs or higher differentiating ability than them. But IRRI‘s NILs almost had no differentiating ability to isolates from japonica rice-growing regions. They can not be used to differentiate races of isolates from japonica ricegrowing regions. Whereas, Chinese NILs can be used to differentiate races of isolates from both japonica ricegrowing regions and indica rice-growing regions. It was concluded that the set of Chinese NILs can be used as international differentials.     

Carbendazim sensitivity in populations of Colletotrichum gloeosporioides complex infecting strawberry and yams in Hubei Province of China

The ascomycete fungus Colletotrichum gloeosporioides is a devastating plant pathogen with a wide host range and worldwide distribution. Carbendazim has been widely used to control anthracnose caused by the C. gloeosporioides complex in China for more than 30 years and resistance to carbendazim has been reported in China. A total of 125 Colletotrichum isolates of strawberry and yam were collected from different geographical regions in Hubei Province, China. Approximately 52.8% of Colletotrichum spp. isolates showed resistance to carbendazim. The isolates tested in this study belong to four species, and the frequencies of resistant isolates differed across Colletotrichum species. Resistant isolates were found in C. siamense and C. fructicola. In contrast, all isolates of C. gloeosporioides and C. aenigma were sensitive to carbendazim. Highly carbendazim-resistant isolates harbored the E198 A mutation in the β-tubulin 2(TUB2) gene, whereas moderately carbendazim-resistant isolates harbored the F200 Y mutation in the TUB2 gene. Carbendazim-sensitive Colletotrichum isolates in this study were not genetically similar enough to form a separate cluster from resistant isolates. The result of this study emphasizes the importance of knowing which Colletotrichum sp. is present, when strategies for disease control are made.     

Inlfuence of Host Shift on Genetic Differentiation of the Oriental Fruit Fly,Bactrocera dorsalis

Invasion of the oriental fruit lfy, Bactrocera dorsalis, into new niches containing different food sources （a process referred to as host shift）, may cause population genetic differentiation and sympatric speciation. To attempt to infer that experimentally, test populations were established by transferring a subset of the original populations, which had been grown on banana for many generations, onto navel orange, and then subculturing the navel orange population and banana population for at least 20 generations. Four pairs of SSR primers with high polymorphism on laboratory strains were used to detect population genetic differentiation. All six tested populations （the 5th, 10th and 15th generations of B. dorsalis fed on banana and navel orange, respectively） were found to have low genetic diversity. Furthermore, the genetic diversity of the navel orange populations was found to decline after being crossed for several generations. Populations initially were deviated from Hardy-Weinberg equilibrium, however, equilibrium was achieved with increasing numbers of generations in both of the host populations. Limited gene lfows were found among the six populations. The Nei’s standard genetic distances between the two host populations of the same generation were initially low, but increased with generation number. Genetic distances between banana and navel orange populations of the same generation were lower than genetic distances between different generations grown on the same host plant. Analysis of molecular distance （AMOVA） results based on generation groups and host groups demonstrated that genetic variation among generations was greater than that between the two host populations. The results indicated that population genetic differentiation occurred after the host shift, albeit at low level. Biogeography and taxonomy of the B. dorsalis complex revealed that speciation of B. dorsalis might be tightly associated with host shift or host specialization of B. dorsalis following dispersal.     

Genetic Variation of Host Populations of Liriomyza sativae Blanchard

In this study, partial sequences of the mitochondrial cytochrome oxidase subunit I （mtDNA-COI） gene and the ribosomal internal transcribed spacer 1 （rDNA-ITS 1） gene, isolated from five artificial populations of Liriomyza sativae （Diptera： Agromyzidae）, were sequenced and compared, to analyze their genetic variation. Analysis of the mtDNA-CO1 gene showed that a low genetic variation was detected among the five populations and only five variable sites were found in the nucleotide sequences. Most of the observed variations that occurred within the populations were because of nucleotide transitions, whereas, the interpopulation variation was because of the differences in haplotype frequencies occurring among the host populations. Analysis of the rDNA-ITS1 gene revealed a small diversity in the five host populations. The trend of genetic differentiation in the host populations was consistent with the preference of L. sativae to the plant hosts.     

Comparative Analysis of Population Genetic Structure in Bemisia tabaci （Gennadius） Biotypes B and Q Based on ISSR Marker

Bemisia tabaci （Gennadius） biotypes B and Q are two invasive biotypes in the species complex. The comparison of the population genetic structure of the two biotypes is of significance to show their invasive mechanism and to their control. The intersimple sequence repeats （ISSR） marker was used to analyze the 16 B-biotype populations and 4 Q-biotype populations worldwide with a Trialeurodes vaporariorum population in Shanxi Province, China, and a B. tabaci non-B/Qbiotype population in Zhejiang Province, China, was used as control populations. The analysis of genetic diversity showed that the diversity indexes of biotype Q including Nei＇s gene diversity index, Shannon informative index, and the percentage of polymorphic loci were higher than those of biotype B. The high genetic diversity of biotype Q might provide the genetic basis for the excellent ecological adaptation. Cluster analysis suggested that the ISSR could not be used in the phylogenetic analysis though it could easily distinguish the biotypes of B. tabaci. The difference of the population genetic structure between the biotype B and the biotype Q exists based on the ISSR marker. Meanwhile, the results suggested that the molecular marker has its limitation in the phylogenetic analysis among the biotypes of B. tabaci.     

Molecular Typing of Aeromonas hydrophila Isolated from Common Carp in Northeast China

A total of 59 isolates of Aeromonas hydrophila were collected from common carp suffering from freshwater fish hemorrhage disease in 13 fishing grounds in the northeast China, and their phenotypic and genetic characteristics were investigated. All of the isolates were identified as A. hydrophila by traditional biochemical method and yielded a 686-bp DNA fragment of the 16S rDNA gene in the PCR experiments. Collected strains were also evaluated for their susceptibility to 17 different antibiotics. The isolates showed an even trend of the resistance and sensitivity to drugs, highly sensitive to antibiotics, such as Levofloxacin, PolymyxinB, Ofloxacin and resistant to antibiotics, such as Bristopen, Lincomycin, Ampicillin, Teicoplanin. Evaluation of genetic diversity was performed on all isolates by molecular typing with enterobacterial repetitive intergenic consensus (ERIC-PCR) method. The results showed that three different types, i.e. type Ⅰ, Ⅱ, and type Ⅲ, were found in 59 isolates and type III accounted for a large proportion of 54.84%. There was no dominant difference between the tendency of the isolates of Heilongjiang Province and Jilin Province in these three types, which showed Ⅲ>Ⅰ>Ⅱ, while the isolates of Liaoning Province showed Ⅲ>Ⅱ>Ⅰ. The percentage of different types in different provinces varied in each other; however, they didn’t show any obvious regional or cluster-specific branches. In conclusion, the ability to distinguish Aeromonas hydrophila strains from diseased common carp with ERIC-PCR would be useful for epidemiological investigation and population genetic analysis of this pathogen in China.     

Population genetic structure of Chinese Puccinia triticina races based on multi-locus sequences

Puccinia triticina, the causal agent of wheat leaf rust, is one of the most devastating rust fungi attacking wheat worldwide. Seventy-six isolates of the wheat leaf rust pathogen from Yunnan, Sichuan, Gansu and Henan provinces, China, were tested on wheat leaf rust differentials and the population structure was analyzed using four presumably neutral partial sequence markers such as elongation factor-1α(EF-1α), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), β-tubulin(TUB) and the second largest RNA polymerase subunit(RPB2). The phenotypic diversity of Yunnan and Sichuan populations was higher than that of Gansu and Henan populations. The four populations were separated into two clusters based on the pathogenic data. A total of 12 single nucleotide polymorphisms(SNPs) and 32 haplotypes were identified among the four sequences. The 32 haplotypes were divided into two clusters in a neighbor-joining tree. Bayesian analyses also identified two clusters. Pairwise FST between populations in different regions were significantly different(P0.05). Analysis of molecular variance(AMOVA) indicated that 68% of the total genetic variation was within populations.     

Population Genetic Analysis of Blumeria graminis f. sp. tritici in Qinghai Province,China

To gain more precise information about molecular genetic variation for wild populations of Blumeria graminis f. sp. tritici from Qinghai Province, China, 38 single-colony isolates were purified from samples collected from Haidong District, Xining City and Hainan Tibetan Autonomous Prefecture in 2010. The virulence of 21 isolates among them was tested at seedling stage on 34 wheat cultivars（lines） carrying known powdery mildew（Pm） resistant genes. The results showed that V1 a, V3 a, V3 c, V3 e, V5 a, V6, V7, V8 and V19 had high virulence frequencies（〉75%）, indicating a wide distribution; and V1 c, V5 b, V12, V13, V16, V21, VXBD, V2＋6, V2＋Mld and V4＋8, with less distribution, appeared to be lower in frequencies（0-20%）. The Nei＇s gene diversity（H）, Shannon＇s information index（I） and the percentage of polymorphic loci（P） were 0.23, 0.35 and 67.65%, respectively, which revealed a virulent diversity. The results from single nucleotide polymorphisms（SNPs） of 38 isolates showed that three housekeeping genes were found to contain a total of 9 SNP sites. 10 haplotypes（H1-H10） were inferred from the concatenated sequences, with 1 haplotype（H1） comprising of over 55% of Qinghai population. Phylogenic analysis did not show obvious geographical subdivision between the isolates. A multilocus haplotype network presented a radial structure, with H1 in the central as an inferred ancestor. Using analysis of molecular variance（AMOVA）, we found 1.63% of the total variation was among populations and 98.37% within populations, with a low fixations index（FST=0.01634, P〈0.05）. This revealed a relatively high genetic diversity but a low genetic divergence in Qinghai population. Moreover, the molecular data on gene flow（Nm=6.32） confirmed the migration of pathogen populations among areas in Qinghai Province.     

Genetic Diversity Based on AIIozyme Alleles of Chinese Cultivated Rice

Genetic diversity was analyzed with 6 632 core rice cultivars selected from 60 282 Chinese rice accessions on the basis of 12 allozyme loci, Pgil, Pgi2, Amp1, Amp2, Amp3, Amp4, Sdhl, Adhl, Estl, Est2, Est5 and Est9, by starch gel electrophoresis. Among the materials examined, 52 alleles at 12 polymorphic loci were identified, which occupied 96.3% of 54 alleles found in cultivated germplasm of O. sativa L. The number of alleles per locus ranged from 2 to 7 with an average of 4.33. The gene diversity （He） each locus varied considerably from 0.017 for Amp4 to 0.583 for Est2 with an average gene diversity （Ht） 0.271, mid Shannon-Wiener index from 0.055 to 0.946 with an average of 0.468. The degree of polymorphism （DP） was in a range from 0.9 to 46.9% with an average of 21.4%. It was found that the genetic diversity in japonica （Keng） subspecies was lower in terms of allele＇s number, Ht and S-W index, being 91.8, 66.2 and 75.7% of indica （Hsien） one, respectively. Significant genetic differentiation between indica and japonica rice has been appeared in the loci Pgil, Amp2, Pgi2, and Est2, with higher average coefficient of genetic differentiation （Gst） 0.635, 0.626, 0.322 and 0.282, respectively. Except less allele number per locus （3.33） for modern cultivars, being 76.9% of landraces, the Ht and S-W index showed in similar between the modem cultivars and the landraces detected. In terms of allozyme, the rice cultivars in the Southwest Plateau and Central China have richer genetic diversity. The present study reveals again that Chinese cultivated rice germplasm has rich genetic diversity, showed by the allozyme allele variation.     

Variations of Single Nucleotide Polymorphism （SNP） of Hordoindoline a Gene （hina） and Its Relationships with Kernel Hardness in 92 North American Barley Varieties （Hordeum vulgare L.）

Grain texture in barley （Hordeum vulgare L.） is an important quality character. The single nucleotide polymorphisms （SNPs） in Hordoindoline a （hina） gene and its relationship with hardness index among North American harley cultivars were investigated. The hina genes in 36 cultivars were sequenced for the SNP analyses, and 17 sets of SNP primers were designed to detect the SNP variations of hina locus in 92 North American barley cultivars. SNP detection indicated that there were four haplotypes in the hina genes of 92 barley cultivars, and haplotype 01 and 02 were shared by 68 and 14 cultivars, respectively, suggesting that there was a very limited diversity in hina genes among North American barley cultivars. Despite the wide range in hardness exists in 92 barley cultivars, however, unlike wheat, where a clear relationship has been demonstrated between a number of SNPs in the wheat hardness genes and quality （soft or hard wheat）, there was no such relationship for barley. The genotypes used in this study demonstrated that there was a low level of polymorphism in hina gene in North American barley cultivars and these polymorphisms had no impact on grain hardness.