Oospore Formation by Phytophthora infestans in Potato Tubers

ABSTRACT The ability of Phytophthora infestans, the causal agent of potato and tomato late blight, to produce oospores in potato tuber tissue was studied in the field and under laboratory conditions. In 1998 and 2000 field experiments, the canopy of potato cvs. Alpha and Mondial, respectively, were coinoculated with A1 + A2 sporangia of the fungus, and the infected tubers collected at harvest were examined for the presence of oospores. In 1998, only 2 of 90 infected tubers had oospores, whereas none of the 90 tubers examined in 2000 had any oospores. In the latter experiment, infected tubers kept in storage up to 12 weeks after harvest had no oospores. Artificial co-inoculations of whole tubers with A1 + A2 sporangia resulted only rarely in the formation of oospores inside the tubers. Co-inoculations of potato tuber discs taken from dormant tubers 0 to 16 weeks after harvest failed to support any oospore production, whereas discs taken from sprouting tubers of >/=18 weeks after harvest allowed oospores to form. Tuber discs showed enhanced oospore formation when treated before inoculation with either sugars, amino acids, casein hydrolysate, beta-sitosterol, or chloroethylphosphonic acid. In contrast, reducing airflow into the petri dishes where potato tuber discs were incubated reduced the number of oospores produced. The number of oospores produced in tuber tissue was lower compared with that in leaf tissue regardless of the origin of isolates used. The data show that the ability of Phytophthora infestans to produce oospores in potato tuber tissue is very limited and increases with tuber aging.     

云南省马铃薯晚疫病菌的交配型、抗药性及生理小种分布的研究

 从云南省陆良县、建水县和南涧县采集2000年秋播的马铃薯晚疫病标本,分离获得124个致病疫霉菌株,连同1999~2000年分离自昆明、曲靖等地的春播马铃薯晚疫病的10个菌株共134个,采用常规技术对这些菌株进行了交配型、抗药性及生理小种的研究。交配型测定结果为:陆良县的18个菌株中有3个为A₂交配型,其余15个菌株为A₁交配型,建水县和南涧县的106个菌株及昆明、曲靖1999~2000年的10个菌株都是A₁交配型。测定了83个菌株对瑞毒霉(metalaxyl)和烯酰吗啉(dimethomorph)的抗药性,结果有12.0%的菌株对瑞毒霉表现抗性,16.9%表现中抗,71.1%表现敏感,其中陆良、昆明和曲靖的抗瑞毒霉菌株的比例大于建水和南涧的抗药性菌株的比例;所测定的83个菌株都对烯酰吗啉表现敏感。生理小种鉴定结果表明,云南晚疫病菌为3.4、0、3和4生理小种,分别占鉴定菌株总数的48.0%、32.5%、15.6%和3.9%。但南涧仅发现3.4和0小种,而昆明有0、3、3.4和4小种,陆良有0、3和3.4小种,可见昆明和陆良地区的晚疫病菌生理小种比南涧地区的生理小种的组成更趋多样。     

Oospore Production of Phytophthora infestans in Potato and Tomato Leaves

ABSTRACT Fungal, host, and environmental factors affecting sexual reproduction of Phytophthora infestans in planta were studied. Intact and detached leaves were coinoculated with sporangia of various combinations of A(1) and A(2) mating-type isolates; leaves were incubated under various conditions, and oospore production was estimated microscopically within whole, clarified leaflets. Some A(1) + A(2) isolate combinations were more reproductive than others, whereas some potato genotypes better supported oospore formation than others. Tomato usually supported more oospore formation than potato. To induce oospore formation, A(1) and A(2) sporangia were usually mixed at a 1:1 ratio. Ratios of 1:19 to 19:1, however, also allowed abundant production of oospores. Optimal temperatures for sexual sporulation ranged from 8 to 15 degrees C, but oospores also were produced at 23 degrees C. Oogonia developed 5 to 6 days after sporangial coinoculation, and oospores developed after 8 to 10 days. Light had little effect on oospore formation in both tomato and potato leaves provided that initial lesions were established under photoperiodic conditions. Although A1 and A(2) sporangia usually were mixed before inoculation on leaves to obtain oospores, we found that discrete A(1) and A(2) lesions produced on opposite sides of the midvein of tomato leaves also induced oospore formation in the midvein and adjacent tissues. Oospores also formed when the two halves of the leaves were cut and separated at 3 days after sporangial coinoculation, which corresponded with the appearance of late blight lesions. The continuous supply of moisture to infected leaves was essential to oospore production. No oospores or oogonia formed in severely diseased plants kept at 50 to 80% relative humidity. Such plants did allow some oospore formation when kept continuously wet for 2 weeks in plastic boxes or tents. Detached leaves floated on water supported the highest sexual sporulation. Under optimal conditions of wetness and temperature, as many as 100 oospores per mm(2) of tissue were observed.     

Phenotypic variation in Nordic populations of Phytophthora infestans in 2003

A total of 743 single-lesion isolates of Phytophthora infestans were collected in summer 2003 from Denmark, Finland, Norway and Sweden. Most of the isolates were tested for mating type, and subsets were tested for sensitivity to fungicides and virulence (host specific pathogenicity). Approximately 60% of the isolates were A1 mating type in each country. Both mating types were present in 40% of the fields where more than one isolate was tested, indicating strong potential for sexual reproduction. The proportion of metalaxyl-resistant isolates dropped to under 15% from the 60% observed in the early 1990s in Norway and Finland, possibly due to lower selection pressure because of decreased use of metalaxyl. Propamocarb-HCl sensitivity remained unchanged in the Nordic countries compared to the situation in 1997–2000 in Finland. Four isolates collected from Finland and Sweden were able to sporulate in the presence of this fungicide at a concentration of 1000 mg L⁻¹. In Norway and Finland the frequencies of virulence factors and pathotypes remained nearly unchanged since the 1990s, but the mean number of virulence factors per isolate increased from 5·6 to 6·3. In Denmark and Sweden virulence factors 2 and especially 6 were more common than in Norway and Finland. In addition, in the Swedish population the frequencies of pathotypes were quite even while in other countries pathotype 1,3,4,7,10,11 was most prevalent.     

黑龙江省马铃薯晚疫病菌交配型及对甲霜灵敏感性测定

马铃薯晚疫病(late blight)是由致病疫霉(Phytophthora infestans)引起的马铃薯生产中最具毁灭性的病害,明确晚疫病菌的交配型及对甲霜灵敏感性状况对制定马铃薯晚疫病防治策略具有重要意义。本研究对2011—2013年采自黑龙江省黑河、齐齐哈尔、绥化等马铃薯主产区的晚疫病菌菌株进行交配型及对甲霜灵敏感性测定,采用对峙培养法对153株晚疫病菌进行交配型检测,结果表明,A1交配型79株,占51.63%,A2交配型74株,占48.37%;采用菌落直径法对143株晚疫病菌进行甲霜灵敏感性测定,结果表明,83.92%表现高抗,8.39%表现中抗,7.69%表现敏感。     

2009年黑龙江和吉林省马铃薯晚疫病菌表型结构研究

为明确2009年黑龙江和吉林两省马铃薯主产区晚疫病菌的表型特征,本研究对所采晚疫病菌的交配型、甲霜灵抗性及生理小种分别进行了测定.结果表明:被测黑龙江(46株)和吉林(48株)两省的菌株均为 A1 交配型.黑龙江省被测菌株甲霜灵高抗、敏感菌株各占50％;吉林省被测菌株中高抗、敏感菌株分别占70.8％、29.2％;两省均未发现中抗菌株.其中采自吉林敦化菌株均为高抗菌株,采自吉林榆树的菌株均为敏感菌株,甲霜灵抗/感比例地区间差异明显.在被测33个黑龙江菌株中发现24个生理小种,而吉林49个菌株中发现19个生理小种,其中9个生理小种两省共有.另外,两省被测菌株中还发现了18株“超级毒力小种”.因此,建议加强种薯监测,防止 A2交配型传入;在生产上使用甲霜灵类药剂防治马铃薯晚疫病应该以菌株的抗性监测数据为依据;进一步加强生理小种监测,合理布局抗病品种.     

Phenotypic and genotypic variation in Michigan populations of Phytophthora infestans from 2008 to 2010

Severe potato and tomato late blight epidemics in Michigan since 2008 prompted characterization of Phytophthora infestans isolates from the region. From eight counties in Michigan, 124 isolates were collected from infected potato and tomato plants from 2008 to 2010 and characterized using ‘classical’ markers and microsatellites. The classical markers included mating type, Gpi allozyme, mitochondrial DNA haplotype, sensitivity to metalaxyl‐M and tuber pathogenicity. All isolates from 2008 to 2010 were A2 mating type and Ia mtDNA haplotype (124 isolates), 105 isolates had Gpi profile 100/122, 17 isolates had the profile 100/100/111 and the remaining two isolates had 100/111/122. Sensitivity to metalaxyl‐M, expressed as EC₅₀ for mycelial growth in vitro, ranged from <0·1 to 91 μg mL^?1, where 95 and 96% of isolates were classified as either sensitive or intermediate in 2008–2009 and 2010 respectively. The metalaxyl‐M sensitivity and dominant Gpi profile were typical of clonal lineage US‐22, first isolated in 2008 in North America from tomato plants. Tuber pathogenicity, characterized as severity of tuber late blight, was also variable among isolates; however, isolates were less aggressive than previous genotypes present in Michigan, such as US‐8. Microsatellites (simple sequence repeats; SSRs) revealed a shift in the population, characterized by two clusters differentiated over time. These results suggested displacement of the US‐8 genotype by US‐22 from 2008 to 2010 in Michigan. Continuous tracking of changes within P. infestans populations provides evidence of genetic shifts due to migration, prompting modification of management strategies based on the phenotypic characteristics of causal genotypes.     

Oospore formation by Phytophthora infestans in host tissue after inoculation with isolates of opposite mating type found in the Netherlands

Samenvatting Uit combinaties van A1- en A2-paringstypen vanPhytophthora infestans werden zowel bij tomaat als bij aardappel oösporen verkregen in de plant. Oösporen werden niet in de bladeren gevormd, maar werden veelvuldig aangetroffen in de epidermis van de stengels. De proeven werden uitgevoerd in de klimaatkamer; de veldsituatie moet nog onderzocht worden.     

Formation and survival of oospores of Phytophthora infestans under natural conditions

Phytophthora infestans is able to produce oospores in leaves of potato and tomato plants after inoculation with a mixture of Al and A2 mating-type isolates. Various conditions for oospore formation were analysed. Under controlled conditions, oospores were produced in potato leaves at temperatures ranging from 5 to 25° C. In leaves of potato cultivar Bintje incubated at 15°C, oogonia and antheridia were observed 6 days after inoculation and thick-walled oospores appeared 3-4 days later. In field experiments oospores were found in leaves and stems of potato cultivars Bintje, Irene and Pimpernel and in leaves, stems and fruits of tomato cultivar Moneymaker within 2 weeks after inoculation. A bioassay was developed to test the survival of oospores in soil under various conditions. To determine whether late-blight infections derived from infectious soil were caused by oospwres, DNA fingerprinting was performed. DNA fingerprint probe RG-57 was suitable for distinguishing asexual progeny from recombinant progeny arising from soil-borne oospores. We demonstrated survival of viable, infectious oospores of P. infestans in soil during the winter of 1992–93. Oospores were not infectious from soil exposed to temperatures of 40°C or higher but in the range 35°C to as low as – 80°C for 48 h, oospores survived.     

Phenotypic and genotypic diversity of Phytophthora infestans populations in Scotland (1995–97)

In a survey of Scottish potato late blight ( Phytophthora infestans ) populations from 1995 to 1997, nearly 500 isolates were collected from over 80 disease outbreaks in commercial potato crops and gardens/allotments. The isolates were characterized by mating type, resistance to the fungicide metalaxyl and almost 300 were examined by DNA-based AFLP fingerprinting. These data were examined alongside cropping details to determine the population structure in the context of existing disease management strategies. A1 and A2 mating type isolates were present in both commercial potato crops and gardens or allotments although they coexisted more frequently in the latter sites. One-fifth of the isolates collected were of the A2 mating type and the frequency was similar over the 3 years and amongst sites. In 1995 the proportions of isolates that were sensitive and resistant to metalaxyl were equal (∼40%) but, over the following 2 years, the frequency of resistant isolates decreased and that of intermediate isolates increased. The mating type response to metalaxyl differed markedly, with 52% of A1 and only 5% of A2 isolates being resistant. Considerable molecular diversity was observed, with over half of the isolates having unique AFLP patterns. Analysis of the molecular and phenotypic data revealed a broad clustering of the population into three groups. Many factors point to an A2 population restricted by its sensitivity to phenylamides. The majority of the A2 isolates were found in a single AFLP group, but the presence of mixed mating type samples, an increasing frequency of isolates of intermediate metalaxyl resistance and the extent of the AFLP diversity suggest occasional sexual recombination, and thus gene flow, between groups.     

Phenotypic variation within a clonal lineage of Phytophthora infestans infecting both tomato and potato in Nicaragua

Late blight caused by Phytophthora infestans (Mont.) de Bary is a constraint to both potato and tomato crops in Nicaragua. The hypothesis that the Nicaraguan population of P. infestans is genotypically and phenotypically diverse and potentially subdivided based on host association was tested. A collection of isolates was analyzed using genotypic markers (microsatellites and mitochondrial DNA haplotype) and phenotypic markers (mating type, virulence, and fungicide sensitivity). The genotypic analysis revealed no polymorphism in 121 of 132 isolates of P. infestans tested. Only the Ia haplotype and the A2 mating type were detected. Most of the tested isolates were resistant to metalaxyl. The virulence testing showed variation among isolates of P. infestans. No evidence was found of population differentiation among potato and tomato isolates of P. infestans based on the genotypic and phenotypic analysis. We conclude that the Nicaraguan population of P. infestans consists of a single clonal lineage (NI-1) which belongs to the A2 mating type and the Ia mitochondrial DNA haplotype. Moreover, based on the markers used, this population of P. infestans does not resemble the population in countries from which potato seed is imported to Nicaragua or the population in neighboring countries. The data presented here indicate that the NI-1 clonal lineage is the primary pathogen on both potato and tomato, and its success on both host species is unique in a South American context.     

Phenotypic and genotypic diversity in Phytophthora infestans on potato in Great Britain, 1995–98

A total of 2691 single-lesion isolates of Phytophthora infestans was established from samples of late-blight disease from 354 commercial and garden/allotment sites in Scotland, England and Wales over four growing seasons, 1995–98. The A2 mating type was rare (3·0% of isolates) and was detected at only 34 sites. In vitro tests of sensitivity to the phenylamide fungicide metalaxyl showed that 316 sites yielded isolates with some insensitivity (resistant and/or intermediate); these were more often commercial sites than garden/allotment sites. Over the four seasons, the frequency of isolates with intermediate fungicide sensitivity increased, while the frequency of resistant phenotypes decreased. Resistant isolates were always of A1 mating type. A subset of 1459 isolates from 326 sites was analysed for molecular diversity. Mitochondrial DNA (mtDNA) haplotype Ia predominated (91·0% of isolates); haplotype IIa was present at 54 sites and both haplotypes at 33 sites. The multilocus RFLP probe RG57 detected 30 fingerprints. Four fingerprints were particularly common (RF002, RF006, RF039 and RF040) and 10 were unique to a single site in a single year. The three commonest fingerprints (RF039 > RF002 > RF006) were of A1 mating type and the fourth (RF040) was A2. RF002 isolates were resistant to the phenylamide metalaxyl and were more common in Scotland than in England and Wales. Small sample sizes limited the usefulness of estimates of diversity. Although approximately half of all sites appeared to be colonized by RF039 genotypes, some sites (both commercial and garden/allotment) showed a higher diversity, having both common and unique genotypes. The genotypic diversity within isolates collected from commercial sites and those from garden/allotment sites were similar. The contributions of sexual reproduction and alternatives to sex to the generation of variation are discussed.     

六省市致病疫霉交配型及其对几种杀菌剂的敏感性

测定了2000-2002年采自四川、重庆、吉林、黑龙江、福建、北京六省(市)致病疫霉菌株的交配型及其对几种杀菌剂的敏感性。结果显示,来自六省市的74个菌株中73株为典型的A1交配型,1株采自四川的菌株为可自育的A1交配型,未发现A2交配型。测定的37株代表菌中,对甲霜灵表现敏感、中抗、高抗的分别占27.0％、27.0％和46.0％。吉林、福建、黑龙江及重庆都存在高抗菌株。研究中还发现甲霜灵可促进部分抗性菌株的菌丝生长。随机抽取15个对甲霜灵表现敏感、中抗和高抗的菌株,测定其对几种杀菌剂的敏感性,结果全部对腈嘧菌酯和烯酰吗啉表现敏感;其中2株对丙酰胺表现敏感,11株表现中抗,2株表现高抗。结果也显示:烯酰吗啉和腈嘧菌酯与甲霜灵之间不存在交互抗性,具有应用潜力。     

Selection for Fungicide Resistance Within a Growing Season in Field Populations of Phytophthora infestans at the Center of Origin

ABSTRACT The central highlands of Mexico should provide an optimal testing ground for evaluating the potential threat of selection for resistance to fungicides in the population of Phytophthora infestans. We evaluated the hypotheses that exposure to the fungicides azoxystrobin, cymoxanil, dimethomorph, fluazinam, mancozeb, metalaxyl, and propamocarb hydrochloride would lead to (i) a shift in the sensitivity distributions (i.e., selection) and (ii) a lower genotypic diversity of the population. We compared populations from unsprayed plots with populations that had been exposed to several applications of each of the fungicides within a single field season. This study provides novel baseline data and shows that the Toluca valley P. infestans population has a wide range of sensitivities to the fungicides fluazinam, cymoxanil, dimethomorph, metalaxyl, and propamocarb. Directional selection toward resistance combined with a reduction in genetic diversity of the P. infestans population was observed only for the fungicide metalaxyl. The results obtained provide direct experimental support for continuing vigilance regarding further introductions of exotic strains of P. infestans into the United States.     

In vivo effects of the fungicide ethaboxam on microtubule integrity in Phytophthora infestans

The effects of ethaboxam (LGC-30473), a novel thiazole carboxamide, on microtubule organization was investigated in Phytophthora infestans (Mont) de Bary (Oomycota) at the light microscope level using standard immunofluorescence and laser scanning confocal microscopy. Hyphal cells were exposed to various concentrations ranging from 0.01 to 1.0 microg ml(-1) of ethaboxam at incubation times of 30, 60 and 120 min. Microtubules were disrupted after exposure to ethaboxam for 30 min at 0.01 microg ml(-1). Similar experimental conditions were used to document the effects of ethaboxam on microtubule integrity in the model fungus Aspergillus nidulans (Eidam) Winter (Ascomycota) and in mammalian mouse 17 clone 1 cells. Under these conditions, there was no evidence to suggest that microtubules in A. nidulans and mouse culture cells were affected by the ethaboxam. These results indicate that ethaboxam is specific for the targeting of microtubule disruption in P. infestans.     

Environmental and Genetic Factors Influencing Self-Fertility in Phytophthora infestans

ABSTRACT Phytophthora infestans is generally regarded as heterothallic-requiring physical proximity of two individuals of different mating type (A1 and A2) for oosporogenesis. Recent reports of limited selfing in young cultures of this oomycete stimulated us to investigate factors contributing to the phenomenon. The ability to produce oospores rapidly (within 2 weeks) in pure, single individual cultures (self-fertility) was tested in 116 individual isolates. The 116 isolates were from geographically diverse locations (16 countries) and were genetically diverse. Mating type and growth medium were the most prominent factors in determining if an isolate would be self-fertile. The majority of A2 isolates (45 of 47 tested) produced oospores when grown on a 50:50 mixture of V8 and rye B medium. In contrast, the majority of A1 isolates (65 of 69 tested) did not produce oospores on this medium. None of the 116 isolates produced oospores when grown on rye B medium (with no V8 juice). Further tests on representative A1 and A2 isolates revealed that oatmeal agar, tomato juice agar, and V8-juice agar all induced the A2 mating type isolate to produce oospores but did not induce the A1 mating type isolate to produce oospores. Calcium carbonate and pH did not alter the self-fertile oospore production in either A1 or A2 mating type isolates. For in vivo tests, the application of fungicide to potato or tomato leaf tissue either before or after inoculation did not stimulate any individual isolate (one A2 and one A1 isolate) to produce oospores in infected tissue. However, in all of the controls for all experiments (in vivo and in vitro), many oospores were produced rapidly if both strains grew in physical proximity.     

Phytophthora infestans Produces Oospores in Fruits and Seeds of Tomato

ABSTRACT Tomato fruits at the mature green stage coinoculated with A1 + A2 sporangia of Phytophthora infestans, the late blight causal fungus, showed abundant oospores in the vascular tissues, pericarp, columella, and placenta. Oospores were also formed on the surface of fruits kept in moisture-saturated atmosphere. Occasionally, oospores were enclosed between the epidermal hairs of the seed coat. In a few seeds, oospores were detected inside the embryo. The data suggest that blighted tomato fruits may carry a large number of oospores, thus making them a threatening source of blight inoculum. Such fruits may also release airborne oosporic inoculum that may introduce recombinant genotypes within a growing season. Although Phytophthora infestans is seedborne in tomato, to our knowledge, this is the first report on the occurrence of oospores in tomato seeds. Whether such tomato seeds produce blighted seedlings remains to be shown.