Pathogenicity and colonization studies on wild–type and auxotrophic isolates of Verticillium albo–atrum from hop

Three fluctuating (M18, M33 and M50) and three progressive (PVl, PV2 and PV3) hop wilt isolates of Verticillium albo–atrum Reinke and Berthold were tested against the hop ( Humulus lupulus L.) cultivars, Fuggle, Wye Challenger and Wye Target. Significant differences in pathogenicity were observed between progressive and fluctuating isolates and although wilt symptoms varied in intensity from season to season, the relative order of pathogenicity and host resistance usually remained the same. Using a detached leaf procedure, differences were observed in the frequency of successful re–isolations of Vertieillium from hop plants infected with wild–type isolates. This procedure is discussed as a possible additional method for distinguishing between progressive and fluctuating hop wilt isolates. Auxotrophic and morphological mutants derived from these six wild–type isolates were tested under glasshouse conditions against hop and the non–selective alternative host Antirrhinum majus L. (cv. Maximum). Although auxotrophic mutants generally showed a reduction in pathogenicity to, and ability to colonize, both hop and Antirrhinum , certain auxotrophs retained near–parental levels of colonization and were therefore suitable for studies on possible parasexual recombination in these hosts.     

Variation for pathogenicity on tomato among parasexual recombinants of Verticillium dahliae

Ten haploid prototrophic recombinant isolates (HPRs) were obtained from each of 15 parasexual crosses between complementary autotrophs derived from nine tomato isolates and one eggplant isolate of V. dahliae , including those identified as race 1 and race 2. These HPRs were tested for pathogenicity to the tomato cultivar Roma which is susceptible to both races. HPRs from a 'selfed' race 2 × race 2 cross were as pathogenic as the wild-type parent. The pathogenicity of HPRs derived from all other crosses was variable, and generally lower than the parental mean. A particularly marked reduction in pathogenicity, compared with the parental mean, was observed for HPRs recovered from two crosses between isolates belonging to different heterokaryon compatibility groups. The results suggest that pathogenicity to cv. Roma is controlled by complex interactions between genes at numerous loci.     

Cultural, molecular and pathogenic variability of Mycosphaerella pinodes and Phoma medicaginis var. pinodella isolates from dried pea (Pisum sativum) in France

The characteristics of 50 isolates of Mycosphaerella pinodes and 17 isolates of Phoma medicaginis var. pinodella , originating from several regions of France where ascochyta blight is prevalent, were investigated using cultural, physiological, molecular and pathogenicity analyses. M. pinodes was distinguished from P. medicaginis var. pinodella on the basis of presence of pseudothecia, a higher proportion of larger, bicellular conidia, compared with the smaller, predominantly unicellular conidia of P. medicaginis var. pinodella , and a slower linear growth rate on agar under a 12-h light regime. RAPD analysis clearly distinguished the two species, which had low intraspecific variability. Although both species gave identical symptoms, they could be distinguished by their incubation period and aggressiveness, respectively, shorter and higher for M. pinodes . Virulence tests gave no definitive evidence for the existence of pathotypes among the M. pinodes isolates. Two unidentified isolates had similar characters to both M. pinodes and P. medicaginis var. pinodella in some features but were distinguished from them by their RAPD patterns.     

球孢白僵菌松毛虫分离株对家蚕的致病力

长期以来对于释放球孢白僵菌防治松毛虫是否会导致或加重家蚕白僵病一直存在较大争议,并同时影响真菌杀虫剂的应用。为科学评价球孢白僵菌松毛虫分离株对家蚕的致病风险,并保证研究结果的代表性和准确性,本研究共使用南方8省（区）的64株松毛虫分离株和4省（区）的12株家蚕分离株对3龄家蚕进行了3次致病性试验。结果表明,除其中1株松毛虫分离株外,不管是松毛虫分离株还是家蚕分离株,对家蚕都有一定致病性,但差异较大。在3×107孢子/mL 的浓度下,30株松毛虫分离株的平均侵染率10.2%极显著低于8株家蚕分离株的89.6%;在1.5×108孢子/mL的浓度下,34株松毛虫分离株对家蚕的平均侵染率36.4%极显著低于6株家蚕分离株的90.6%;松毛虫分离株在1.5×108孢子/mL浓度下的侵染率极显著高于3×107孢子/mL下的侵染率,而家蚕分离株在两种浓度下无显著性差异。在1.5×108孢子/mL的浓度下4株对家蚕致病力较强的松毛虫分离株的LT50为7.45～8.20 d,平均7.83 d,比6株家蚕分离株LT50（3.43～3.72 d）的平均数3.62 d多4.21 d。根据毒力较强的家蚕分离株B1和B5与毒力最弱和最强的松毛虫分离株D26和D22的致死浓度的比较,家蚕分离株对家蚕的毒力是松毛虫分离株的132～15733倍;根据他们的致死剂量的比较,家蚕分离株的毒力是松毛虫分离株的43～2201倍。这一对比数据充分表明,尽管不同的松毛虫分离株对家蚕的毒力不同,但所有的松毛虫分离株都具有明显的寄主专化性。综合分析表明,我国南方使用白僵菌杀虫剂防治松毛虫在家蚕中诱发流行病的风险很小。     

Barrage Zone Formation Between Vegetatively Incompatible Fusarium graminearum (Gibberella zeae) Isolates

ABSTRACT Vegetative compatibility has been used to assess the population biology of many fungal plant pathogens. However, for many species, including Fusarium graminearum, this has meant making auxotrophic mutants to force heterokaryon formation. A method was developed to observe barrage zones of thick, raised mycelium at the junctions of vegetatively incompatible F. graminearum isolates. The appearance of the barrage zones was influenced by the growth medium and the light. Barrage zones on V8 agar were thicker and better defined than those on potato dextrose agar, Spezieller Nahrstoffarmer agar, and water agar. The addition of ground wheat kernels to V8 agar enhanced barrage zone formation. Incubating the cultures under constant light at 2,150 lx produced more distinct barrage zones than constant light at 3,400 lx, constant darkness, or ambient room light. Forty-three F. graminearum isolates from 34 vegetative compatibility groups, determined previously using nit auxotrophic mutants, were paired in all combinations using these optimized conditions. Isolates in different vegetative compatibility groups typically formed distinct, thick barrage zones at their junctions. Pairs of isolates in the same vegetative compatibility group had a very slight or no visible reaction, or rarely, a distinct "line gap" of sparse mycelium. Subcultures from the same isolate typically had no visible reaction at their colony junctions; however, subcultures from some isolates had thin, slight barrage zones. This method was used to identify the proportion of each of four F. graminearum isolates from infected barley spikes in the field, inoculated previously with a mixture of conidia from these four isolates. Barrage zone formation represents a rapid method to screen vegetative compatibility groups in F. graminearum and may be useful for other Fusarium species.     

Cultural Characteristics, Pathogenicity and Vegetative Compatibility of Fusarium udum Isolates from Pigeonpea (Cajanus cajan (L.) Millsp.) in Kenya

Seventy-nine single-spore isolates of Fusarium udum, the causal agent of wilt disease of pigeonpea, from Kenya, India and Malawi were characterized according to their cultural characteristics, pathogenicity and vegetative compatibility group (VCG). The isolates exhibited high variation in pathogenicity on a wilt-susceptible pigeonpea variety, and in mycelial growth and sporulation on potato dextrose agar medium. The 79 isolates were categorized into two virulence groups, two groups of radial mycelial growth and four groups of sporulation. Radial mycelial growth showed a moderate negative correlation (r = –0.40; P = 0.01) with sporulation. However, mycelial growth and sporulation had no correlation with virulence. Pairings between complementary nitrate non-utilizing (nit) mutants of F. udum generated on chlorate containing minimal medium revealed that all the isolates belonged to a single VCG (VCG 1) with two subgroups, VCG 1 I and VCG 1 II. Vegetative compatibility was independent of cultural characteristics and pathogenicity. This is the first report of vegetative compatibility in F. udum.     

Persistence and fitness of carbendazim- and dicarboximide-resistant isolates of Monilinia fructicola (Wint.) Honey in flowers, shoots and fruit of stone fruit

Flowers on detached peach shoots and ripe fruit were inoculated under controlled conditions in order to estimate the competitive ability of Monilinia fructicola isolates sensitive and resistant to carbendazim and dicarboximide fungicides. Isolates varied considerably, but there was no consistent relationship between carbendazim resistance and competitive ability; there was, however, evidence of reduced competitive ability in the single dicarboximide-resistant and the dual dicarboximide/high carbendazim-resistant isolate examined. The ability to produce conidia on twig cankers inoculated in late spring 1989 was retained under field conditions by all sensitive and resistant isolates for at least 1 year. Dicarboximide-resistant isolates produced fewer conidia than the carbendazim-resistant and sensitive isolates on cankers. The production of conidia on mummified fruit inoculated in February 1990 decreased in the field during the winter and the following spring, but some conidia were produced by all isolates. Measures of pathogenicity, virulence and fitness for all isolates were similar to the original values after survival for 1 year. The evidence presented in this paper supports field observations that carbendazim-resistant isolates are likely to persist permanently in the M. fructicola population, whereas dicarboximide-resistant isolates are more likely to decline unless fungicide selection pressure is maintained.     

Vegetative compatibility and heterokaryon stability in Fusarium oxysporum f.sp. radicis-lycopersici from Italy

Fusarium crown and root rot, caused by Fusarium oxysporum f.sp. radicis-lycopersici ( Forl ), is one of the most destructive soilborne diseases of tomato in Italy. Chlorate-resistant, nitrate-nonutilizing ( nit ) mutants were used to determine vegetative compatibility among 191 isolates of Forl collected in five geographic regions (Calabria, Emilia-Romagna, Liguria, Sardinia, Sicily) in Italy. The isolates were assigned to five vegetative compatibility groups (VCGs): 65 isolates to VCG 0090; 99 to VCG 0091; 23 to VCG 0092; two to VCG 0093; and two to VCG 0096. The population structure of Forl in Italy is similar to that reported for Israel, and differs from that found in North Atlantic European countries, where VCG 0094 is predominant. The stability of prototrophic heterokaryons originating from hyphal anastomosis between compatible complementary nit mutants was assessed through conidial analysis and mycelial mass transfer. Most monoconidial cultures (84%) recovered from 117 prototrophic heterokaryons were nit mutants, indicating that heterokaryons generally do not proliferate well through conidiation; most of the 177 prototrophic heterokaryons examined were unstable, and only 9% sustained prototrophic growth through the tenth mycelial transfer upon subculturing. The prototrophic growth is proposed to be maintained through restoration of the heterokaryotic state by continual anastomosis between adjacent homokaryotic hyphae. Since heterokaryosis is a prerequisite for parasexual recombination, we speculate that this mechanism is unlikely to play a major role in generating the VCG diversity found among Forl or other strains of F. oxysporum.     

Phytopathogenic, morphological, genetic and molecular characterization of a Verticillium dahliae population from Crete, Greece

A population of 84?V. dahliae isolates mainly originating from Crete, Greece, was characterized in terms of pathogenicity and virulence on different hosts, in parallel with morphological/physiological characterization, vegetative compatibility grouping and mating type determination. Tomato race 2 was found to have supplanted race 1 and was more virulent on a tomato-susceptible cultivar than race 1. Using a differential host classification system which tests pathogenicity to tomato, eggplant, sweet pepper and turnip, 59 isolates were assigned to tomato, 19 to eggplant, one to sweet pepper and five to tomato-sweet pepper pathogenicity groups. All isolates from Crete fell into VCG subgroups 2A, 2B and 4B, while a remarkably high incidence of bridging isolates (compatible with two or more VCGs) was recorded. The tomato-sweet pepper pathogenicity group was morphologically quite distinct from the others, while conidial length and pigment intensity were discriminatory parameters among VCGs 2A, 2B and 4B. PCR-based molecular marker Tr1/Tr2 was reliable in race prediction among tomato-pathogenic isolates, except for members of VCG 4B, while the application of markers Tm5/Tm7 and 35-1/35-2 was highly successful for tomato-pathogenic isolates. E10 marker was related to VCG 2B, rather than to pathogenicity groups. A single nucleotide polymorphism in the ITS2 region, and two novel molecular markers, M1 and M2, proved useful for the fast and accurate determination of major VCGs 2A, 2B and 4B, and can be used for high-throughput population analyses in future studies. The mating type was unrelated to VCG classification and probably does not control heterokaryon incompatibility in V. dahliae.     

Vegetative compatibility groups and parasexual segregation in Colletotrichum acutatum isolates infecting different hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.     

Phylogenetic analysis based on ITS sequences and conditions affecting the type of conidial germination of Bipolaris oryzae

One taxonomic characteristic of Bipolaris species is the bipolar germination of conidia, but conidia of Bipolaris oryzae , the causal pathogen of brown spot in rice, are regularly observed to show intercalary germination, a characteristic of Drechslera species. The effect of selection, culture media and culture age on type of conidial germination was determined for three brown spot isolates from Cavinti, San Pablo and Palawan in the Philippines, obtained from infected leaves showing typical disease symptoms. Based on the analyses of their ITS1, ITS2 and 5·8S rDNA nucleotide sequences, the local isolates were clearly identified as B. oryzae . Selection for colonies of the three isolates derived from single conidia with either bipolar or intercalary germination had no effect on the number of spores showing bipolar germination in subsequent cultures. Germination on seven different culture media was tested; of these, rabbit food agar and water agar increased the percentage of bipolar germination of conidia, although this varied between isolates. Incubation of the cultures of all three isolates for longer periods prior to harvesting conidia increased the percentage of bipolar-germinating conidia from c . 40 to c . 90% with 5-day-old and 30-day-old cultures, respectively.     

Relationship between the virulence of hop isolates of Verticillium albo-atrum and their in vitro secretion of cell-wall degrading enzymes

The in vitro production of extracellular polygalacturonase, pectin lyase, and cellulase by hop isolates of Verticillium albo-atrum was studied. A total of 24 isolates belonging to the two pathotypes commonly described as fluctuating or progressive, according to the symptoms produced in certain susceptible hop cultivars, were grown in liquid shake cultures. The media used contained glucose, pectin or acetone-extracted hop tissue as a carbon source. The production of all three enzymes was significantly correlated with pathotype when the medium contained hop tissue. Pectin lyase production was also correlated with pathotype when pectin was used as the carbon source. The influence of carbon source on enzyme output and possible reasons for some exceptions to the correlations observed are discussed.     

稻瘟病菌营养体亲和配对筛选及其亲和能力遗传

 在白米玫瑰红培养基上对江苏省的稻瘟病菌Magnaporthe grisea(Hebert) Barr.不同小种间营养体亲和菌株配对组合进行了筛选。当不同小种的菌株对峙接种,在21±1℃、黑暗中培养18 d后,部分配对组合的菌落交界处出现一条肉眼明显可见的菌丝融合带,且具有稳定的重复性。出现菌丝融合带的配对菌株在玻璃纸上对峙微培养2~3 d后,镜检观察到菌丝融合现象普遍存在;未出现明显菌丝融合带的菌株组合经对峙微培养,菌丝融合现象未见或较少。从ZA₄₉、ZG₁、ZF₁和ZD₁ 4个小种的12个菌株中筛选出3对营养体亲和性强的小种间菌株配对组合。以所选菌株为亲本建立单分生孢子无性系,测定营养体亲和能力在单孢后代的遗传。结果是,营养体亲和能力在单孢后代遗传不稳定,与另一亲本的单孢株配对产生融合带的百分率为53%~74%之间。进一步研究发现,来自同一个分生孢子不同细胞的单芽管株与配对菌株的营养体亲和能力相同;亲和能力在单芽管株的单孢株后代继续发生分离。     

Vegetative Compatibility Groups in Cercospora kikuchii, the Causal Agent of Cercospora Leaf Blight and Purple Seed Stain in Soybean

ABSTRACT Nitrogen nonutilizing (Nit) mutants were used to assess vegetative compatibility of 58 isolates of Cercospora kikuchii, 55 of which were isolated from soybean plants in Louisiana. Two isolates were vegetatively self-incompatible. Of 56 self-compatible isolates, 16 were assigned to six multimember vegetative compatibility groups (VCGs), 01 to 06, with 2 or 3 isolates in each VCG. The other 40 isolates each belonged to a distinct VCG. All six multimember VCGs contained isolates from different soy bean cultivars, and three included isolates from different locations. Only one of six multimember VCGs included isolates both from soybean leaves and seed, while the other five included isolates from only leaves or seed. The likelihood of tissue specificity or preference was discussed. All isolates and tested Nit mutants produced cercosporin on potato dextrose agar under light. Significantly different amounts of cercosporin were produced among wild-type isolates, and two Nit mutants produced significantly more cercosporin than their wild-type counterparts. All isolates produced typical Cercospora leaf blight symptoms on soybean plants in greenhouse pathogenicity tests.     

江苏麦类禾谷镰刀菌变异性的研究

1980～1981年选取代表江苏不同地区引致麦类赤霉病的禾谷镰刀菌野生型和培养型菌株17个,并人工移植1～3次后,观察培养性状的变化;同时分别接种感病小麦品种“矮秆早”,观察致病性的变化,以分析病菌的变异性。结果证明:江苏极大多数禾谷镰刀菌菌株的野生型经人工移植三次后,无论是培养性状和致病力都发生了改变。一般表现力生长速度减慢,产孢数量变少,产孢速度变慢,分生孢子变小,分生孢子分隔数减少,不易产生分生孢子或不产孢,不易形成子囊壳或不产生子囊壳而产生粘孢团,以及致病力减弱。但是,只有少数菌株的变异特别明显。以上结果说明禾谷镰刀菌菌株中存在有变异现象,但不同菌株并不一致,培养性状变异与致病力强弱变异间的关系也可能因菌株而异。至于禾谷镰刀菌在无性培养中发生变异的原因还有待进一步研究。     

啤酒花根癌土壤杆菌生物型的鉴定及生物防治的初步探讨

啤酒花根癌病在我国发生普遍,是影响啤酒花生产的重要病害。从啤酒花主要生产基地新疆、山东、浙江等地分离到61个菌株。通过培养性状、染色反应,以及14～15个菌株的生理生化性状和12个菌株致病性的测定,确定啤酒花根癌病病原菌为根癌土壤杆菌Agrobacteria tumefaciens(E.F.Smith &Jownsend)Conn属生物型1、病原菌菌株对K84(Agrobacterium radiobacter)菌株的细菌素敏感,各菌株间敏感性的强弱不一。盆裁番茄和啤酒花初步生防试验表明,用K84与敏感性强的菌株按1:1比例混合接种,番茄和啤酒花都不产生瘿瘤;而敏感性弱的菌株需用3:1才能防止瘿瘤的产生。     

New Media for Increasing Sporulation and Germination of Phaeoisariopsis Griseola Conidia

The slow growth rate of the fungus Phaeoisariopsis griseola and the availability of a homogeneous highly concentrated inoculum is an important constraint for pathogenicity or virulence studies, where plant inoculations are needed. Therefore, the purpose of this paper was to evaluate the effect of supplementing culture media with Amaranthus cruentus seed meal on fungal growth and sporulation of isolates of P. griseola belonging to the Mesoamerican and Andean groups. The amendment of PDA or V8 media with A. cruentus seed meal resulted in a considerable increase in the number of conidia and also in their capacity to germinate; this depended mostly on the stage of maturity of conidia. Mesoamerican and Andean isolates produced a different number of conidia when cultured in vitro. Furthermore, while in Mesoamerican isolates a second degree polynomial represented the relationship between number of conidia and amount of A. cruentus supplementation, in Andean isolates the relationship was linear. It seems that either one or several of the nutritional factors provided by A. cruentus contributed to the increased production of conidia and their development, resulting in faster development of the disease and an earlier appearance of symptoms. Therefore, for cultural studies, especially for inoculum production and for pathogenicity evaluations, supplementation of the media with A. cruentus seed meal proved to be a good alternative.     

芝麻棒孢叶斑病病原菌鉴定与致病力比较

 为明确芝麻叶斑病病原菌及其致病力,本研究以河南不同地区芝麻上分离所得叶斑病分离物为研究对象,通过形态观察和分子鉴定探讨这些分离物的分类属性,并且进行离体叶片和活体茎杆接菌,进一步探究了不同菌株的致病力差异。结果显示分离所得的6株芝麻叶斑病分离物的菌落形态、生长速率和产孢量存在差异,分生孢子多数呈倒棍棒状,具隔膜,6个菌株的ITS序列与GenBank中Corynespora cassiicola的一致性达99%以上,结合形态特征与ITS序列将其鉴定为山扁豆生棒孢。室内人工接菌芝麻发现,这6个菌株的致病力存在显著的差异;在芝麻叶片上,20180909-03属强致病力菌株,20180821-01、20180909-05和20180824-01属中等致病力菌株,20180904-02-02和SF1-1属弱致病力菌株;在芝麻茎杆上,20180824-01致病力最强,其次是20180821-01、20180909-03和20180909-05,SF1-1和20180904-02-02致病力最弱。研究结果证明山扁豆生棒孢不同菌株在芝麻上的致病力存在明显的分化。     

芝麻棒孢叶斑病病原菌鉴定与致病力比较

 为明确芝麻叶斑病病原菌及其致病力,本研究以河南不同地区芝麻上分离所得叶斑病分离物为研究对象,通过形态观察和分子鉴定探讨这些分离物的分类属性,并且进行离体叶片和活体茎杆接菌,进一步探究了不同菌株的致病力差异。结果显示分离所得的6株芝麻叶斑病分离物的菌落形态、生长速率和产孢量存在差异,分生孢子多数呈倒棍棒状,具隔膜,6个菌株的ITS序列与GenBank中Corynespora cassiicola的一致性达99%以上,结合形态特征与ITS序列将其鉴定为山扁豆生棒孢。室内人工接菌芝麻发现,这6个菌株的致病力存在显著的差异;在芝麻叶片上,20180909-03属强致病力菌株,20180821-01、20180909-05和20180824-01属中等致病力菌株,20180904-02-02和SF1-1属弱致病力菌株;在芝麻茎杆上,20180824-01致病力最强,其次是20180821-01、20180909-03和20180909-05,SF1-1和20180904-02-02致病力最弱。研究结果证明山扁豆生棒孢不同菌株在芝麻上的致病力存在明显的分化。     

Growth rates of, and mycelial interactions between, isolates of Crinipellis perniciosa from cocoa

Growth of 87 single-basidiospore isolates of Crinipellis perniciosa , derived from witches' brooms on cocoa in 10 localities throughout South America and the Caribbean, was examined at 25°C on five agar media (Czapek-Dox, prune extract, potato-dextrose, V8 and carboxymethylcellulose). Interactions (designated intermingling or mutually antagonistic) between paired mycelia of 64 isolates were determined on V8 plates.
Six somatic-compatibility groups were identified comprising isolates from; (1) Pichilingue and Rio Palenque in Ecuador, Chigorodo and Manizales in Colombia; (2) Sucua (Ecuador); (3) Manaus (Brazil); (4) Ouro Preto (Brazil); (5) Castanhal (Brazil); (6) Trinidad and Tobago. This geographical separation of isolates was supported by results of the growth tests; growth of isolates within each compatibility group differed from other groups on at least one of the five media.
Separation of isolates by these methods did not conflict with previous results from tests of pathogenicity and could be useful in selecting isolates for screening cocoa lines for resistance to C. perniciosa.