Production and Germination of Conidia of Trichoderma stromaticum, a Mycoparasite of Crinipellis perniciosa on Cacao

ABSTRACT Growth characteristics of the fungus Trichoderma stromaticum, a mycoparasite on the mycelium and fruiting bodies of Crinipellis perniciosa, the causal agent of witches'-broom disease of cacao, were evaluated under controlled environmental conditions. The ability of T. stromaticum to produce conidia and germinate on dry brooms was evaluated at three constant temperatures (20, 25, and 30 degrees C) and two constant relative humidities (75 and 100%). T. stromaticum produced abundant conidia on brooms at 100% relative humidity and incubation temperatures of 20 and 25 degrees C, but none at 30 degrees C. Sporulation of T. stromaticum was not observed at 75% relative humidity at any temperature. At 100% relative humidity and either at 20 or 25 degrees C, treatment of brooms with T. stromaticum suppressed C. perniciosa within 7 days. In contrast, at 30 degrees C, treatment with T. stromaticum had no effect on the pathogen in brooms maintained at either 75 or 100% relative humidity. Mycelium of C. perniciosa grew from brooms at all temperatures at 100% relative humidity. Conidial germination on broom tissue approximated 80% at temperatures from 20 to 30 degrees C. Results suggest that applying T. stromaticum under high-moisture conditions when the air temperature is below 30 degrees C may enhance the establishment of this mycoparasite in cacao plantations.     

Canopy-microclimate effects on the antagonism between Trichoderma stromaticum and Moniliophthora perniciosa in shaded cacao

The collective impact of several environmental factors on the biocontrol activity of Trichoderma stromaticum ( Ts ) against Moniliophthora perniciosa ( Mp ), the cause of cacao witches' broom disease, was assessed under field conditions of shaded cacao ( Theobroma cacao ) in south-eastern Bahia, Brazil. Biocontrol experiments were performed adjacent to an automated weather station, with sensors and Ts -treated brooms placed at different canopy heights. Sporulation occurred at the same dates for all Ts isolates, but in different quantities. Broom moisture >30%, air temperature of approximately 23 ± 3°C, relative humidity >90%, solar radiation intensities <0·12 KW m⁻² and wind speed near zero were the key environmental parameters that preceded Ts sporulation events. A multiple logistic regression indicated that these weather variables combined were capable of distinguishing sporulation from non-sporulation events, with a significant effect of wind speed. Analyses of environmental factors at ground level indicated similar pre-sporulation conditions, with a soil moisture content above a threshold of 0·34 m³ m⁻³ preceding all sporulation events. The sporulation of five selected Ts isolates was compared at four different canopy heights. Isolates responded differently to weather variation in terms of sporulation and antagonism to Mp at different canopy levels, indicating that different microclimates are established along the vertical profile of a shaded cacao plantation. The potential of these findings for development of predictive mathematical models and disease-management approaches is discussed.     

Genetic variation in populations of the cacao wilt pathogen, Ceratocystis cacaofunesta

Ceratocystis cacaofunesta (=  Ceratocystis fimbriata ) causes a lethal wilt disease of cacao ( Theobroma cacao ) in Latin America. Polymorphic microsatellite markers, (CAT)₅ nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS-rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.     

Comparison of Crinipellis perniciosa isolates from Brazil by ERIC repetitive element sequence-based PCR genomic fingerprinting

Fifty isolates of Crinipellis perniciosa originating from Theobroma cacao , Heteropterys acutifolia and Solanum lycocarpum , from six states within Brazil, were characterized through ERIC-PCR, representing the first application of this method for molecular characterization within C. perniciosa . Phenetic analysis of banding patterns revealed a separation of isolates on the basis of host of origin, with T. cacao -derived isolates showing only a 0·2 similarity level to a cluster comprising the isolates from H. acutifolia and S. lycocarpum . Considerable intraspecific variability was observed within C. perniciosa isolates from T. cacao , with distinct groups observed correlating with geographical origin. Given that a number of isolates from T. cacao from the Amazon region grouped with isolates from Bahia state, this work discusses the possibility that current C. perniciosa populations pathogenic on T. cacao in Bahia originated from the Amazon region, rather than from alternative host plants.     

Pathogenic variability amongst isolates of Crinipellis perniciosa from cocoa (Theobroma cacao)

Dry witches' brooms from cocoa were imported from various areas within Bolivia, Brazil, Colombia, Ecuador, Trinidad and Venezuela. Basidiocarps of Crinipellis perniciosa were induced to form on these brooms and seedlings of different types of cocoa were inoculated with basidiospores either on the hypocotyl or cotyledon bud. Host reactions were assessed mainly by recording stem base swelling and broom development at the cotyledon node (hypocotyl inoculations) or the extent of swelling and branching of shoots (cotyledon bud inoculations).
Results from 30 experiments indicated considerable diversity amongst isolates in inducing disease symptoms, but suggested that two groups or populations of C. perniciosa exist on cultivated cocoa. One group (A), comprising isolates from Bolivia and Pichilingue (Ecuador) and most isolates tested from Colombia, induced severe symptoms on cocoa with Scavina 6 as one parent; the other group (B), comprising isolates from Brazil, Trinidad and Venezuela, did not. Within these groupings variants could be further distinguished by particular host reactions. Isolates from Ecuador, especially from the Oriente, a centre of diversity for Theobroma cacao, showed a range of pathogenicity comparable to that found amongst isolates from cultivated cocoa over a much wider area.     

Molecular Fingerprinting Suggests Two Primary Outbreaks of Witches' Broom Disease (Crinipellis perniciosa) of Theobroma cacao in Bahia, Brazil

Crinipellis perniciosa (Stahel) Singer is the causal agent of witches' broom disease in the Sterculiaceae, Solanaceae, and Bixaceae families. The disease is endemic to the Brazilian Amazon, and was first reported infecting Theobroma cacao (cocoa) in the State of Bahia, Brazil, in 1989. Random amplified polymorphic DNA (RAPD) analyses were performed on 46 isolates of C. perniciosa from cocoa that were collected from 15 counties in Bahia and the Brazilian Amazon. A total of 258 RAPD loci from 20 primers and three mixed primers were analyzed. Of these loci, 108 (42%) were polymorphic, with an average of 4.7 polymorphic loci per primer produced. Genetic similarities were estimated using Nei and Li's index and UPGMA clustering. Bootstrap analysis divided the phenogram into four significantly different clusters: two groups contained isolates from Ariquemes and from Ouro Preto, Rondônia, and the other two separated the isolates from Bahia into two major groups of C. perniciosa, classified as Group 1 (G1) and Group 2 (G2). The two groups of isolates from Bahia differed for their genetic similarity with the isolates from the Brazilian Amazon. The geographic distribution of the groups in Bahia suggests two independent focal points of introduction. Ongoing programs to screen for resistant cocoa genotypes should consider both groups of isolates.     

Ceratocystis wilt of cacao-a disease of increasing importance

ABSTRACT Ceratocystis cacaofunesta (formerly C. fimbriata) causes a lethal wilt disease of cacao (Theobroma cacao) in the Caribbean and Central and South America. Recent studies employing phylogenetics, intersterility, and host range separate the cacao pathogen from other strains of the C. fimbriata complex. Ceratocystis wilt has been managed through genetic resistance, but the disease is an emerging problem in Bahia, Brazil, where it was recently introduced. Genetic studies indicate that populations of the fungus in Costa Rica, Colombia, and Bahia may have been introduced on cacao cuttings; whereas populations in Rond?nia, Brazil, and western Ecuador appear to be native. The fungal genotype present in Bahia is similar to those found in Rond?nia and may have been introduced on propagative material with witches' broom resistance.     

黑龙江省水稻纹枯病菌的致病力分化与AFLP分析

为了明确黑龙江省水稻纹枯病菌遗传多样性,为水稻抗病育种和水稻纹枯病的综合防治提供依据。本文对采自13个水稻种植地区的29个水稻纹枯病菌菌株进行了致病力测定和AFLP分析。结果表明9对AFLP引物对供试菌株扩增出396条带,其中多态性带187条,占总扩增带数的47.22%。黑龙江省水稻纹枯病菌的遗传距离变化在0.50~0.92之间,平均为0.71,群体遗传多样性较为丰富。UPGMA法可以将供试菌株分成4个AFLP聚类组群(Ⅰ、Ⅱ、Ⅲ和Ⅳ),相同地理来源的菌株基本上聚集在同一组群内,表明AFLP类群划分与菌株的地理来源有较强的相关性。黑龙江省水稻纹枯病菌致病性分化较为明显,并且AFLP类群划分与菌株的致病性鉴定之间存在一定相关性。     

VCG and AFLP analyses identify the same groups in the causal agents of mango malformation in Brazil

The causal agents of mango malformation disease in Brazil are a new Fusarium lineage in the Gibberella fujikuroi species complex and Fusarium sterilihyphosum; however information on the genetic and geographical diversity of these pathogens in Brazil is missing. Vegetative compatibility group (VCG) and amplified fragment length polymorphism (AFLP) analyses were used to measure the genetic diversity within these populations. Both techniques identified the same genetic groups. Six VCG and AFLP groups were identified amongst isolates of the new lineage from Brazil. FB-VCG 1/AFLP I was the most widespread group, found in seven of the 13 sites sampled. The second most frequent group was recovered from three sites. The remaining four groups were recovered from single-sites. We think that this lineage represents a genetically and geographically diverse indigenous population that reproduces clonally. In F. sterilihyphosum, group FS-VCG 1/AFLP VII was found at three sites in the southeast region of Brazil. FS-VCG 2/AFLP VIII contained isolates from South Africa but not from Brazil. Fusarium mangiferae isolates from India and South Africa formed one group, while isolates from Egypt and the USA formed a second group. F. sterilihyphosum at present is represented by a small population that might have been introduced only once into a restricted area. The clonal nature of the observed populations suggests that these fungi either occur naturally on indigenous hosts and have jumped to the introduced mango host (introduced in Brazil) or that they originated with mango and went through a severe population bottleneck when they were introduced to Brazil from India or Southeast Asia.     

Population structure and migration of the witches’ broom pathogen Moniliophthora perniciosa from cacao and cultivated and wild solanaceous hosts in southeastern Brazil

Moniliophthora perniciosa, causal agent of witches’ broom disease in cacao plantations in South America and the Caribbean Islands, has co‐evolved with its host cacao, but the pathogen has also emerged in many solanaceous hosts in Brazil, including economically important food crops and wild species. This study was carried out to: (i) determine the existence of host subpopulations of M. perniciosa in Brazil; (ii) estimate gene and genotypic diversity of M. perniciosa host subpopulations infecting solanaceous hosts in southeastern Bahia and Minas Gerais states, Brazil; and (iii) estimate the amount and directionality of historical migration of M. perniciosa subpopulations. Up to 203 M. perniciosa isolates collected from solanaceous hosts with symptoms from Bahia and Minas Gerais states in Brazil and from Theobroma spp. (cacao) and Herrania spp. were characterized with 11 microsatellite markers. Factorial correspondence analyses, minimum‐spanning network and Bayesian clustering revealed genetic clusters associated with their host of origin. Significant subpopulation differentiation was evident (Φ_ST = 0.30, P ≤ 0.05) among M. perniciosa host subpopulations. Most of the multilocus microsatellite genotypes (MLMGs) were host‐specific, with few MLMGs shared among subpopulations. Pairwise comparisons among M. perniciosa host subpopulations were significant, except between jurubeba (Solanum paniculatum) and cultivated solanaceous subpopulations. The combined analyses rejected the null hypothesis that M. perniciosa in Brazil is a single genetic population not structured by host. These findings support a scenario of introduction and subsequent adaptation to solanaceous hosts that should be taken into consideration to improve mitigation and management of M. perniciosa.     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

Spatial genetic structure and dispersal of the cacao pathogen Moniliophthora perniciosa in the Brazilian Amazon

Moniliophthora perniciosa is the causal agent of witches’ broom in Theobroma cacao (cacao). Three biotypes of M. perniciosa are recognized, differing in host specificity, with two causing symptoms on cacao or Solanaceae species (C‐ and S‐biotypes), and the third found growing endophytically on lianas (L‐biotype). The objectives of this study were to clarify the genetic relationship between the three biotypes, and to identify those regions in the Brazilian Amazon with the greatest genetic diversity for the C‐biotype. Phylogenetic reconstruction based on the rRNA ITS regions showed that the C‐ and S‐biotypes formed a well‐supported clade separated from the L‐biotype. Analysis of 131 isolates genotyped at 11 microsatellite loci found that S‐ and especially L‐biotypes showed a higher genetic diversity. A significant spatial genetic structure was detected for the C‐biotype populations in Amazonia for up to 137 km, suggesting ‘isolation by distance’ mode of dispersal. However, in regions containing extensive cacao plantings, C‐biotype populations were essentially ‘clonal’, as evidenced by high frequency of repeated multilocus genotypes. Among the Amazonian C‐biotype populations, Acre and West Amazon displayed the largest genotypic diversity and might be part of the centre of diversity of the fungus. The pathogen dispersal may have followed the direction of river flow downstream from Acre, Rondônia and West Amazon eastward to the rest of the Amazon valley, where cacao is not endemic. The Bahia population exhibited the lowest genotypic diversity, but high allele richness, suggesting multiple invasions, with origin assigned to Rondônia and West Amazon, possibly through isolates from the Lower Amazon population.     

Biodiversity and biogeography of the cacao (Theobroma cacao) pathogen Moniliophthora roreri in tropical America

Moniliophthora roreri , the cause of moniliasis or frosty pod rot, occurs on the neotropical rainforest genera Theobroma and Herrania . While this basidiomycete has had devastating effects on the cacao tree ( T. cacao ) in tropical America, where it is confined, little is known of its biogeography and intraspecific genetic variability. Here, AFLP and ISSR profiles of 94 isolates of M. roreri from across its geographic range in Central/South America were analyzed. The study provided limited evidence to support the hypothesis that M. roreri is capable of sexual reproduction. The highest levels of genetic diversity occurred in Colombia and not in Ecuador as originally believed. The fungus was broadly divided into five genetic groups. Two of these have a wide geographic range: Bolívar group (north of Santander in Colombia, eastern Venezuela, peripheral Ecuador, Peru), and Co-West group (western Colombia, central Ecuador, Central America). The other groups are all apparently endemic to Colombia (Co-East and Co-Central groups) or north-western Ecuador ( Gileri group). We speculate that central/north-eastern Colombia may represent the centre of origin for M. roreri . Sequence data from the internal transcribed spacer region of the nuclear rDNA repeat were congruent with the AFLP/ISSR results, dividing M. roreri into two broad groups: the Orientalis group, comprising most isolates from the Co-East, Co-Central and Bolívar groups, and the Occidentalis group, comprising isolates from the Co-West and Gileri groups. The spread of M. roreri into new areas and countries mediated by human activity is discussed.     

Analysis of molecular diversity in <Emphasis Type="Italic">Crinipellis perniciosa</Emphasis> with AFLP markers

Crinipellis perniciosa causes a serious disease of cacao known as witches broom (WB). Heritable resistance to witches broom has been used in cacao improvement programs. SCA6 and SCA12 are highly resistant and are the most commonly used parents in the breeding schemes. However, SCA hybrids are not resistant to witches broom in all production areas. Presumably, different populations of C. perniciosa cause these variable responses. Amplified fragment length polymorphism (AFLP) markers were used to assess variation and population structure in this pathogen. We examined 40 isolates of C. perniciosa and one isolate of Melanotus subcuneiformis. Nine of 64 primer pairs produced consistent and informative DNA amplification, and were used to screen all isolates. Fifteen haplotypes (AFLP fingerprints) were detected with 186 polymorphic markers. Cluster analysis grouped isolates of the C biotype (pathogenic on cacao) from Bolivia, Brazil, Ecuador and Trinidad together in a major cluster that was distinct from isolates of the S biotype (pathogenic on solanaceous hosts) and M. subcuneiformis. Isolates of the C biotype were divided further into well supported, country-specific groups. Segregation of AFLP alleles was not observed among basidiospore isolates from the same basidiome, broom, tree or field, supporting previous reports that the fungus did not outcross. The results corroborated prior conclusions that C. perniciosa was probably introduced into the Bahia state of Brazil from the Amazon basin. Representative isolates from the genetically distinct groups that were revealed will be used to examine pathogenic specialization in C. perniciosa and differential responses that have been reported in SCA6-derived germplasm.     

Random amplified polymorphic DNA (RAPD) analysis of Crinipellis perniciosa isolates from different hosts

DNA sequence polymorphisms among isolates of Crinipellis perniciosa , causing witches' broom disease in several genera and species in the Sterculiaceae, Solanaceae and Bixaceae, were revealed by random amplified polymorphic DNA (RAPD) banding patterns. Comparisons were made among isolates from Theobroma cacao (cultivars SCA6 and SVB), T. grandiflorum, T. obovatum, T. subincanum, Herrania sp. (all collected near Belém, Brazil, at three plots within two kilometres of each other), and Bixa orellana and Solanum rugosum (150 and 1440 km from Belém, respectively). Genetic similarities, as determined by Sorensen's coefficient ( Sc_ij ; fraction of shared bands), ranged from 1·00 ( T. cacao SCA6 and T. grandiflorum ) to 0·56 ( S. rugosum and T. subincanum). Results indicate that proximity is more important than host species for determining the genetic relationships among isolates, that there were two independent sources of inoculum at the site near Belém, and that the isolate from B. orellana is most closely related to the isolate from T. cacao SVB. Comparisons were also made among multispore cultures from different basidiocarps on the same T. cacao SVB broom, as well as monospore cultures from the same S. rugosum basidiocarp. Banding patterns were similar among basidiocarps on the same broom, but differences were detected among monospore cultures from the same basidiocarp.     

Relationships Between Black Pod and Witches'-Broom Diseases in Theobroma cacao

ABSTRACT Field observations were conducted from 1998 to 2001 at the International Cocoa Genebank, Trinidad, to evaluate 57 cacao clones for resistance to black pod (BP) and witches'-broom (WB) diseases (caused by Phytophthora sp. and Crinipellis perniciosa, respectively). Each month ripe pods were harvested and the number of healthy and diseased was recorded. The number of brooms on vegetative shoots was recorded three times a year on selected branches. Twenty-three clones showed less than 10% of infection for both BP and WB on pods. Among those, eight clones showed an absence of brooms on the observed branches: IMC 6, MAN 15/60 [BRA], PA 67 [PER], PA 195 [PER], PA 218 [PER], PA 296 [PER], PA 303 [PER], and POUND 32/A [POU]. Broad-sense heritability was estimated at 0.38 and 0.57 for WB disease on pods and shoots, respectively, and at 0.51 for BP disease. Genetic correlation between WB disease on pods and on shoots was low and estimated at 0.39, whereas the correlation between WB and BP diseases on pods was 0.48. To choose putative parents for breeding schemes, it is suggested that clones are first assessed for their level of resistance to WB on shoots, and the most promising individuals are screened for BP with a detached pods test. Further studies are needed to confirm whether the level of resistance to WB on pods can be predicted using an early test on seedlings.     

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches?? broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70?M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.     

水稻品种构成对稻曲病菌遗传结构影响的初步研究

由稻曲病菌（Ustilaginoidea virens）引起的病害是当前限制我国水稻优质高产的重要病害之一。家畜食用被稻曲病菌厚垣孢子污染的谷物可导致中毒。近20年来，稻曲病防治主要以喷施化学农药为主。研究品种布局对稻曲病菌群体结构的影响，有助于了解病害的发生规律及提出有效的病害防治策略。本文初步分析了稻曲病菌群体结构与水稻品种构成的关系。     

Diversity of Meloidogyne incognita populations from cotton and aggressiveness to Gossypium spp. accessions

The root-knot nematode (RKN) Meloidogyne incognita is the main nematode causing losses to the cotton crop in Brazil. In order to implement control strategies within integrated management, an accurate identification of the nematode populations prevailing in the cotton production areas is necessary. This study aimed to assess the genetic variability and aggressiveness of RKN populations from cotton production areas in Bahia state, Brazil. All populations were characterized biochemically and molecularly and identified as M. incognita. RAPD and AFLP markers detected 44% of polymorphic fragments among the 13 populations of this species. The 10 M. incognita populations collected in Bahia presented 33.7% of diversity when compared to each other, and 25% when the population from Barreiras (the most polymorphic) was excluded. This polymorphism increased when populations from other Brazilian states were included. The aggressiveness and virulence among populations from Bahia towards different cotton accessions (susceptible/resistant) was also studied. None of the populations showed virulence against the moderately resistant (Clevewilt 6, Wild Mexican Jack Jones and LA 887) and highly resistant (CIR1348 and M-315 RNR) cultivars. Two M. incognita populations from Barreiras were the most aggressive, reaching reproduction factors of 539 and 218, respectively, in the susceptible cultivar FiberMax 966. The most aggressive population (8) was also the most genetically divergent in phylogenetic analyses. These results demonstrate that diversity of M. incognita populations from cotton farms in Bahia is not related to virulence against resistant accessions, which suggests that cultivars containing one or two resistance genes with good agronomic characteristics could be used in infested commercial areas in Bahia state, Brazil.     

影响水稻纹枯病菌营养体亲和性分化的因子

 为了探明影响水稻纹枯病菌营养体亲和性分化的因子,将水稻纹枯病菌菌株cx-2在不同水稻品种继代接种,并在含不同杀菌剂、具不同pH及不同温度条件的PDA平板上继代培养,用对峙法测定继代菌株与原始接种菌株的营养体亲和性。此外,对营养体亲和性分化菌株与原始菌株的AFLP指纹图谱进行比较。结果显示,在供试的30个水稻品种上连续接种4次后,从9个水稻品种中分离出与原始菌株营养体不亲和的菌株。菌株在不同pH值的PDA平板继代培养4次后,在pH偏碱性端(pH 10、pH 11)开始出现营养体亲和性分化的菌株。在不同农药和温度条件下继代培养10次的菌株中没有分离到营养体亲和性分化的菌株。营养体亲和性分化菌株与原始菌株的AFLP指纹图谱没有差异。