Genetic Analysis of Metalaxyl Insensitivity Loci in Phytophthora infestans Using Linked DNA Markers

ABSTRACT Previous studies indicated that incompletely dominant loci determine insensitivity by oomycetes to phenylamide fungicides such as metalaxyl. To compare the bases of insensitivity in different strains of the late blight pathogen, Phytophthora infestans, crosses were performed between sensitive isolates and isolates from Mexico, the Netherlands, and the United Kingdom that displayed varying levels of insensitivity. Segregation analyses indicated that metalaxyl insensitivity was determined primarily by one locus in each isolate, and that two of the isolates were heterozygous and the other homozygous for the insensitive allele. Metalaxyl insensitivity was also affected by the segregation of additional loci of minor effect. DNA markers linked to insensitivity were obtained by bulked segregant analysis using random amplified polymorphic DNA (RAPD) markers and the Dutch and Mexican crosses. By studying the linkage relationships between these markers and the insensitivity in each cross by RAPD or restriction fragment length polymorphism analysis, it appeared that the same chromosomal locus conferred insensitivity in the Mexican and Dutch isolates. However, a gene at a different chromosomal position was responsible for insensitivity in the British isolate.     

Dispersal of Phytophthora nicotianae on tomatoes grown by nutrient film technique in a greenhouse

Tomatoes were grown in a greenhouse by nutrient film technique (NFT). They were inoculated withPhytophthora nicotianae either by direct inoculation of roots or by adding fungal spores to a container with recirculating nutrient solution. In the first case, the resulting epidemic seemed to be polycyclic, in the second case monocyclic. In both cases, inoculum freely circulated through the NFT system and was present in the nutrient solutions for at least 6 days after inoculation.Samenvatting Kastomaten werden gekweekt met behulp van voedingsfilmtechniek. Zij werden geinoculeerd metPhytophthora nicotianae hetzij door directe besmetting van wortels, hetzij door een sporensuspensie toe te voegen aan het voorraadvat met voedingsoplossing. In het eerste geval leek een polycyclische epidemie te volgen. In het tweede geval ontstond een monomcyclische epidemie. In beide gevallen werd schimmelinoculum door het gehele gotensysteem rondgepompt. Inoculum bleef gedurende tenminste 6 dagen na inoculatie aantoonbaar in de voedingsoplossing.     

Studies on the inheritance of resistance to metalaxyl in Phytophthora infestans

Single-oospore progeny from matings between field isolates of Phytophthora infestans either highly resistant or sensitive to metalaxyl were analysed for fungicide sensitivity in vitro, mating type and, in some cases, allozyme variation at the locus for glucosephosphate isomerase ( GPI-1. ) In each cross the majority of first-generation (F₁) progeny showed intermediate sensitivity to metalaxyl. Frequency distributions were skewed towards sensitivity and a few progeny were either wholly sensitive or resistant phenotypes. Allozyme analysis of F₁ progeny from a cross between parents of Dutch and Mexican origin showed that c. 10% were selfs of both parents and of parental phenotype for metalaxyl sensitivity. The selfs of the A2 but not the Al parent segregated for mating type. Two backcross generations to the metalaxyl-resistant Dutch parent gave unexpected phenotypic frequency distributions and aberrant ratios for genotypes at the GPI-I locus.
Progeny of another backcross between an F₁ isolate of intermediate sensitivity and its sensitive Egyptian parent gave a 1:1 ratio for sensitive: intermediate phenotypes. F₂ progeny from a related sibmating between intermediate phenotypes segregated in a ratio close to 1:2:1 for sensitive: intermediate: resistant phenotypes. Segregation was also observed among sexual progeny of an intermediate self-fertile isolate from the backcross generation but not among progeny of a resistant self-fertile phenotype. Mating type segregated in both cases.
These data are consistent with resistance to metalaxyl in P. infestans being governed by a single nuclear locus exhibiting incomplete dominance. This hypothesis is discussed in relation to the incidence of resistance to metalaxyl in natural populations of P. infestans.     

马铃薯晚疫病菌对甲霜灵抗性机制的初步研究

 选取对甲霜灵敏感、中抗和高抗的马铃薯晚疫病菌(Phytophthora infestans)菌株各2株,采用电导率法和同位素掺入法,研究了甲霜灵对马铃薯晚疫病菌不同抗药性水平菌株细胞膜通透性和RNA聚合酶活性的影响,借助液相色谱分析方法检测了不同抗药性水平菌株对甲霜灵的降解作用。结果表明:甲霜灵对马铃薯晚疫病菌不同抗药性水平菌株的细胞膜通透性没有明显影响;随着甲霜灵浓度提高,敏感菌株的RNA聚合酶活性受到显著抑制,10μg/mL甲霜灵对敏感菌株RNA聚合酶活性的抑制率达到40%以上,而10和100μg/mL甲霜灵对高抗菌株的RNA聚合酶活性只表现轻微的抑制作用,抑制作用分别小于8%和21%,甲霜灵对中抗菌株RNA聚合酶活性的抑制作用介于对敏感菌株和高抗菌株的抑制作用之间。抗性菌株对甲霜灵有一定的降解作用,培养30d后高抗菌株HL105和SC1的降解率分别达到13.46%和16.14%。     

Stability of resistance to Phytophthora infestans in potato: an international evaluation

Ten institutions in nine countries joined together to test the stability of resistance of 14 potato genotypes to the oomycete pathogen Phytophthora infestans in three separate trials. Seven of the genotypes were tested in one trial involving seven locations, and all 14 were tested in two subsequent trials, each involving eight locations. Stability of resistance was tested with nonparametric tests and with an additive main effects and multiplicative interaction (AMMI) model. Overall, resistance to P. infestans was robust; resistant genotypes were consistently resistant in all locations and trials. The nonparametric analysis indicated that specific genotypes were basically stable across sites for resistance. In trial 3, the Z statistic for overall stability was significant at 0·05%, indicating a significant level of interaction across the trial, but there were no significant interactions for specific genotypes in this trial. The genotype by environment (G × E) effect of the AMMI model was highly significant in both trials, but the mean square of G × E was less than 10% of the genotype effect in each trial. The first two principal components (PCA1 and PCA2) of the AMMI analyses together explained 75 and 80% of the interaction effects in trials 2 and 3, respectively. Based on both nonparametric and AMMI analyses, Ecuador and Argentina were locations of relatively high interaction effects for both trials 2 and 3, although in Ecuador this interaction was not associated with any particular potato genotype. Other locations also had high interaction effects, but these occurred in only one trial. The genotypes Chata Blanca and, to a lesser extent, Torridon were relatively unstable in trials 2 and 3, but in the case of Torridon, resistant, this did not represent a significant loss of resistance.     

Effects of fertilizers and plant strengtheners on the susceptibility of tomatoes to single and mixed isolates of Phytophthora infestans

Three organic fertilizers, Horn meal, i.e., BioFeed Basis and Bio-ILSA, and three plant strengtheners, i.e., Alfalfa extract, PEN and QUALITY were compared to chemical fertilizer (NPK) application and the synthetic inducer BABA (DL-3-amino-butyric acid) for reactions of leaf disks of six different tomato cultivars to three isolates of Phytophthora infestans. Late blight severity was significantly reduced on plants fertilized with Bio-ILSA and BioFeed Basis compared to plants fertilized with Horn meal and chemical fertilizer. There were no interactions between fertilizers and isolates or between fertilizers and cultivars. All plant strengtheners significantly reduced susceptibility of all tomato cultivars. However, effects were isolate and tomato cultivar specific. The reductions in area under the disease progress curve compared to the water control for the different tomato cultivars and isolates were 23–78%, 21–77%, 17–66%, and 37–100% for Alfalfa extract, PEN, QUALITY and BABA, respectively. Similar, but somewhat smaller reductions were observed for sporulation capacity. The plant strengtheners were more effective in controlling disease on plants challenged with isolate mixtures than on plants challenged with single isolates. BABA performed significantly better than the plant strengtheners in 65% (i.e., 34 out of 54) of the tested cultivar-isolate combinations, when single isolates were used. When two isolates were mixed, the percentage was reduced to 45% (25 out of 54 cases) and to 33%, (6 out of 18 cases) with a mixture of three isolates.     

Nine years of practical experience with phenylamide resistance in Phytophthora infestans in the Netherlands

Resistance to phenylamide fungicides in thePhytophthora infestans population in the Netherlands decreased from a level of 77% of the samples containing resistant strains in 1980 to 0% in 1986 with decreasing use of metalaxyl, the only phenylamide fungicide registered for use against late blight in this country. Use of metalaxyl decreased because the fungicide alone and combinations of metalaxyl with preventive late blight fungicides were not commercially available from 1981 through the first part of the potato growing season of 1984. When metalaxyl was made available again in August 1984 in a mixture with mancozeb and from 1985 in a mixture with almost full rates of maneb and fentinacetate its use remained limited because growers were advised to apply the combination only two times per season in critical situations only to control the severe late blight epidemics of 1984 and 1985. Moreover application on seed potato crops was not allowed. In 1986 the combination was hardly used because the mild epidemic was easily controlled by preventive compounds only.In 1987 and 1988 phenylamide resistance increased to 16 and 42%, respectively, inherent to a general use of the combination in both years in most potato growing regions of up to two applications to combat a very severe epidemic.In lots of seed potatoes harvested in 1988 the percentage of blighted tubers yielding resistant isolates significantly differed from 13% for regions where seed potatoes are predominantly grown for table and processing crops to 41% for a region where predominantly starch potatoes are grown, both as seed and production crops. The more intensive use of metalaxyl combinations in the latter area through the years as compared to the other areas is assumed to be responsible for the higher incidence of resistance.In most years application of the combination induced a significant shift in the population to resistance as is indicated by the higher frequency at which resistant isolates were present in leaf samples taken from treated fields or tuber samples from the harvested crop.The strategy adopted by Dutch potato growers to combat phenylamide resistance in late blight, involving limited use of metalaxyl mixtures to a maximum of two times in critical situations only, has kept up to now resistance in the population at a level at which metalaxyl still significantly contributes to the efficacy of the combination in controlling the disease. Never-theless the observed increase in resistance in the population of 1988 after two consecutive late blight years indicates that this may change if severe epidemics occur each year.     

Stability of partial resistance in potato cultivars exposed to aggressive strains of Phytophthora infestans

Potato cultivars were evaluated for their resistance responses to aggressive strains of Phytophthora infestans in field and laboratory experiments. Analysis of variance revealed differential cultivar-by-isolate interactions for both foliar and tuber blight resistance. Differential responses occur as revealed by specific susceptibilities of cultivars to certain pathogen genotypes and changing rank order. In general, severity of late blight epidemics as observed in the haulms did not correlate well with foliar blight resistance ratings as presented in the National List of Recommended Potato Varieties. No significant correlation was found between tuber blight incidence under field conditions and the tuber blight rating in the National List. Also, there was no relation between the field and laboratory tuber blight resistance assessments. A significant association was demonstrated between late blight infection in the foliage and tuber blight incidence under field conditions. The presence of differential interaction, independent of R-gene-based resistance, indicates some adaptation of P. infestans to partial resistance and consequently adverse effects on the stability and durability of partial resistance to potato late blight.     

Evaluation of foliar resistance in potato to Phytophthora infestans based on an international field trial network

During the period 2000–03, local potato cultivars in Estonia, Latvia, Lithuania, Poland and Denmark were tested for foliar resistance to Phytophthora infestans (late blight) in an international field trial network. Four standard cultivars were included in the trials: Sava, Oleva, Danva and Kuras. Primary disease-assessment data were entered into a common database, and parameters from the disease progress curves were calculated and made available on interactive web pages. A regression model, using relative area under disease progress curve (RAUDPC) values for cv. Oleva as a reference, was developed for the estimation of 1–9 scale values, where 1 = most susceptible. Standard deviations for the estimated 1–9 scale values and a nonparametric rank stability analysis of RAUDPC were used to evaluate the stability of resistance of the cultivars. Overall, the results showed stability of resistance for cvs Sava, Oleva and Danva, but not for Kuras. Use of the Internet-based Web-Blight service in this study facilitated comparison of results among countries for the level and stability of resistance. The estimated 1–9 scale values were similar to, or slightly lower than, those from official cultivar lists or from the European Cultivated Potato Database, especially for the more resistant cultivars. Possible reasons for discrepancies from different sources and locations are discussed. It is concluded that RAUPDC and the derived 1–9 scale values are useful for ranking cultivars for resistance to P. infestans , but this information is not detailed enough for use in a decision support system for late blight control.     

Survival of Phytophthora infestans in Surface Water

ABSTRACT Coverless petri dishes with water suspensions of sporangia and zoospores of Phytophthora infestans were embedded in sandy soil in eastern Washington in July and October 2001 and July 2002 to quantify longevity of spores in water under natural conditions. Effects of solar radiation intensity, presence of soil in petri dishes (15 g per dish), and a 2-h chill period on survival of isolates of clonal lineages US-8 and US-11 were investigated. Spores in water suspensions survived 0 to 16 days under nonshaded conditions and 2 to 20 days under shaded conditions. Mean spore survival significantly increased from 1.7 to 5.8 days when soil was added to the water. Maximum survival time of spores in water without soil exposed to direct sunlight was 2 to 3 days in July and 6 to 8 days in October. Mean duration of survival did not differ significantly between chilled and nonchilled sporangia, but significantly fewer chilled spores survived for extended periods than that of nonchilled spores. Spores of US-11 and US-8 isolates did not differ in mean duration of survival, but significantly greater numbers of sporangia of US-8 survived than did sporangia of US-11 in one of three trials.     

A review of biocontrol agents in controlling late blight of potatoes and tomatoes caused by Phytophthora infestans and the underlying mechanisms

Phytophthora infestans causes late blight on potatoes and tomatoes, which has a significant economic impact on agriculture. The management of late blight has been largely dependent on the application of synthetic fungicides, which is not an ultimate solution for sustainable agriculture and environmental safety. Biocontrol strategies are expected to be alternative methods to the conventional chemicals in controlling plant diseases in the integrated pest management (IPM) programs. Well-studied biocontrol agents against Phytophthora infestans include fungi, oomycetes, bacteria, and compounds produced by these antagonists, in addition to certain bioactive metabolites produced by plants. Laboratory and glasshouse experiments suggest a potential for using biocontrol in practical late blight disease management. However, the transition of biocontrol to field applications is problematic for the moment, due to low and variable efficacies. In this review, we provide a comprehensive summary on these biocontrol strategies and the underlying corresponding mechanisms. To give a more intuitive understanding of the promising biocontrol agents against Phytophthora infestans in agricultural systems, we discuss the utilizations, modes of action and future potentials of these antagonists based on their taxonomic classifications. To achieve a goal of best possible results produced by biocontrol agents, it is suggested to work on field trials, strain modifications, formulations, regulations, and optimizations of application. Combined biocontrol agents having different modes of action or biological adaptation traits may be used to strengthen the biocontrol efficacy. More importantly, biological control agents should be applied in the coordination of other existing and forthcoming methods in the IPM programs. © 2023 Society of Chemical Industry.     

Aggressiveness of Phytophthora infestans and phenotypic analysis of resistance in wild Petota accessions in Ecuador

The aggressiveness of four Phytophthora infestans isolates collected from wild and cultivated potato species (sect. Petota ) and the level of resistance of nine Petota species were assessed in the highland tropics of Ecuador. For this, isolates of P. infestans were inoculated on whole plants of Petota species in the field and net house and six epidemiological components – infection frequency (IF), incubation period (IP), latent period (LP), lesion size (LS), lesion growth rate (LGR), and relative area under the lesion expansion curve (RAULEC) – were measured during a single infection cycle. Additionally, host specificity was determined by testing for a significant host by pathogen interaction using the same components. The results showed significant differences among isolates of the EC-1 clonal lineage for IP, IF, and RAULEC. Significant differences among isolates were not found for the other components measured. There were significant differences in resistance among the accessions of Petota hosts tested. RAULEC, LGR, LP, and LS were in general more adequate in differentiating among the more resistant and more susceptible accessions but the importance of each component varied with host species. There was slight and inconsistent evidence for the existence of host specificity in some isolates of Petota hosts. IP was the only component for which a significant host by isolate interaction was observed and in most cases the isolates had the greatest aggressiveness on their hosts of origin.     

Population changes in Phytophthora infestans in Taiwan associated with the appearance of resistance to metalaxyl

In recent years, late blight, caused by Phytophthora infestans (Mont) De Bary, has increased in severity in many parts of the world, and this has been associated with migrations which have introduced new, arguably more aggressive, populations of the pathogen. In Taiwan, late blight has been endemic on outdoor tomato crops grown in the highlands since the early 1900s, but recent epidemics have been more damaging. To ascertain the present status of the Taiwanese population of P infestans, 139 isolates of the pathogen collected and maintained by the Asian Vegetable Research and Development Center (AVRDC) were characterized using mating type, metalaxyl sensitivity, allozyme genotype, mitochondrial haplotype and RFLP fingerprinting. Up to 1997, all isolates were found to belong to the old clonal lineage of P infestans (US-1 and variants), but in isolates from 1998 a new genotype appeared, and by 2000 this had apparently completely displaced the old population. This new genotype was an A1 mating type and has the dilocus allozyme genotype 100/100/111, 100/100 for the loci coding for glucose-6-phosphate isomerase and peptidase, respectively. These characters, together with RG57 fingerprinting, indicated that these isolates belonged to the US-11 clonal lineage, a minority (11%) being a previously unreported variant of US-11. Whereas metalaxyl-resistant isolates were not detected in the old population, 96% of the new genotypes proved resistant, with the remainder being intermediate in sensitivity. It may be inferred from this sudden, marked change in the characteristics of the Taiwanese P infestans that a new population of the pathogen was introduced around 1997-98 and that this may well have already been metalaxyl-resistant when it arrived, although a role for in situ selection cannot be excluded.     

Diversity of Phytophthora infestans from Poland

A collection of 96 Polish isolates of Phytophthora infestans sampled in the years 2006, 2008 and 2009 were analysed using phenotypic and genotypic markers. Mating type, virulence, resistance to metalaxyl, mitochondrial haplotype and polymorphism at 12 simple sequence repeat (SSR) loci were determined. The majority of isolates were of the A1 mating type, mitochondrial haplotype Ia and sensitive to metalaxyl. Virulence factors against potato R genes R1, R3, R4, R7, R10 and R11 were present in most isolates. Genotyping using SSR markers revealed high genetic diversity within the Polish P. infestans population. Amongst the 96 isolates 66 unique genotypes were identified, 49 of which were observed only in single isolates. Eight isolates of the genotype 13_A2 lineage that has been reported in other parts of Europe were also found in Poland. The implications of these results are discussed.     

Transcriptome profile of acibenzolar-S-methyl-induced genes in tomato suggests a complex polygenic effect on resistance to Phytophthora infestans

Induced resistance by chemicals such as acibenzolar-S-methyl -ASM (commercialized as Actigard by Syngenta Inc) mimics the biological activation of systemic acquired resistance (SAR). ASM takes the place of salicylic acid (SA) in the SAR signal pathway inducing the same molecular markers and range of resistance. The goal of our work was to understand the downstream molecular events by which ASM confers resistance to Phytophthora infestans in tomatoes. To accomplish this goal we assayed gene expression in ASM-treated plants using a microarray with more than 12,000 tomato ESTs. As many as 300 genes were responsive to ASM. Of these, 117 were detected in most of the biological replications. Basal defense associated genes as well as SAR and disease resistance genes (R-like) involved in induced resistance and effector-triggered immunity were highly expressed. We attempted to determine the phenotype of 13 of these genes by virus induced gene silencing (VIGS). These 13 genes were selected on the basis of previous implication in plant defense response and by reliability of induction by ASM. VIGS was partially successful for three of the 13 genes, but this partial silencing did not lead to a significant reduction in the effect of ASM. The ethylene pathway was also activated in response to ASM, but a tomato mutant not responsive to ethylene remained responsive to ASM. It seems most likely that the ASM effect is complex and polygenic, depending on the effect of several genes.     

Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island

Isolates of Phytophthora erythroseptica , causal agent of pink rot of potato, were obtained from diseased tubers collected in Prince Edward Island (PEI), Canada. In an in vitro assay of field and single-zoospore isolates, all isolates of the pathogen from PEI were sensitive to metalaxyl (EC₅₀ < 0·5  µ g mL⁻¹), unlike moderately or highly resistant reference isolates obtained from Maine, USA. Allozyme-banding patterns at the glucose-6-phosphate isomerase ( Gpi ) locus were identical (91/91) for all isolates of P. erythroseptica examined from PEI and Maine, but could be used to distinguish isolates of P. erythroseptica from local isolates of Phytophthora infestans . Inoculation of potato tubers from plants treated with foliar applications of metalaxyl in the field indicated that compounds inhibitory to metalaxyl-sensitive isolates of P. erythroseptica were present in the periderm, even after 4 months' storage. By contrast, moderately or highly resistant isolates of the pathogen caused significantly ( P  ≤ 0·05) more disease than sensitive isolates in tubers from plants treated with metalaxyl. The effectiveness of metalaxyl in controlling pink rot in a particular region will depend on resistance levels of local populations of P. erythroseptica . Preventing the development and spread of resistant strains of P. erythroseptica will be critical in maintaining metalaxyl as an effective chemical for control of this pathogen.