Metalaxyl-sensitive Isolates Research Articles

High Temporal Variability in Late Blight Pathogen Diversity, Virulence, and Fungicide Resistance in Potato Breeding Fields: Results from a Long-Term Monitoring Study.

Long-term site-specific studies describing changes in the phenotypic variability of Phytophthora infestans populations allow quantitative predictions of pathogen spread and possible outbreaks of epidemics, and provide key input for regional resistance breeding programs. Late blight samples were collected from potato (Solanum tuberosum) breeding fields in Estonia during a twelve-year study period between 2001 and 2014. In total, 207 isolates were assessed for mating type and 235 isolates for metalaxyl resistance and 251 isolates for virulence factors. The frequency of mating types strongly fluctuated across the years, whereas the later period of 2010–2014 was dominated by the A2 mating. Despite fluctuations, both mating types were recorded in the same fields in most years, indicating sustained sexual reproduction of P. infestans with oospore production. Metalaxyl-resistant and intermediately resistant strains dominated in the first years of study, but with the progression of the study, metalaxyl-sensitive isolates became dominant, reaching up to 88%. Racial diversity, characterized by normalized Shannon diversity index decreased in time, varying from 1.00 in 2003 to 0.43 in 2013. The frequency of several virulence factors changed in a time-dependent manner, with R2 increasing and R6, R8, and R9 decreasing in time. Potato cultivar resistance background did not influence the frequency of P. infestans mating type, response to metalaxyl, and racial diversity. However, the diversity index decreased in time among isolates collected from resistant and susceptible cultivars, and remained at a high level in moderately resistant cultivars. These data demonstrate major time-dependent changes in racial diversity, fungicide resistance, and virulence factors in P. infestans, consistent with alterations in the control strategies and popularity of potato cultivars with different resistance.

Highly Diverse Phytophthora infestans Populations Infecting Potato Crops in Pskov Region, North-West Russia.

There is limited understanding of the genetic variability in Phytophthora infestans in the major potato cultivation region of north-western Russia, where potato is grown primarily by small households with limited chemical treatment of late blight. In this study, the mating type, sensitivity to metalaxyl, and genotype and population genetic diversity (based on 12 simple sequence repeat (SSR) markers) of 238 isolates of P. infestans from the Pskov region during the years 2010–2013 were characterized. The aim was to examine the population structure, phenotypic and genotypic diversity, and the prevalent reproductive mode of P. infestans, as well as the influence of the location, time, and agricultural management practices on the pathogen population. The frequency of the A2 mating was stable over the four seasons and ranged from 33 to 48% of the sampled population. Both mating types occurred simultaneously in 90% of studied fields, suggesting the presence of sexual reproduction and oospore production in P. infestans in the Pskov region. Metalaxyl-sensitive isolates prevailed in all four years (72%), however, significantly fewer sensitive isolates were found in samples from large-scale conventional fields. A total of 50 alleles were detected in the 141 P. infestans isolates analyzed for genetic diversity. Amongst the 83 SSR multilocus genotypes (MLGs) detected, 65% were unique and the number of MLGs varied between locations from 3 to 20. These results, together with the high genotypic diversity observed in all the locations and the lack of significance of linkage disequilibrium, suggest that sexual recombination is likely responsible for the unique MLGs and the high genetic diversity found in the Pskov region population, resembling those of north-eastern European populations.

Chickpea Seed Rot and Damping-Off Caused by Metalaxyl-Resistant Pythium ultimum and Its Management with Ethaboxam.

Metalaxyl and its isomer mefenoxam have been the primary fungicides used as seed treatments in managing Pythium seed rot and damping-off of chickpea (Cicer arietinum). However, outbreaks of seed rot and damping-off of metalaxyl-treated chickpea seeds were found in the dryland agriculture regions of southeastern Washington and northern Idaho. Pythium spp. isolated from rotten seeds and associated soils showed high levels of resistance to metalaxyl. Large proportions (31 to 91%) of Pythium isolates resistant to metalaxyl were detected in areas where severe chickpea damping-off occurred and were observed in commercial chickpea fields over several years. All metalaxyl-resistant (MR) isolates were identified as Pythium ultimum var. ultimum. The metalaxyl resistance trait measured by EC50 values was stable over 10 generations in the absence of metalaxyl, and no observable fitness costs were associated with metalaxyl resistance. Under controlled conditions, metalaxyl treatments failed to protect chickpea seeds from seed rot and damping-off after inoculation with MR Pythium isolates. In culture, ethaboxam inhibited mycelial growth of both MR and metalaxyl-sensitive isolates. Greenhouse and field tests showed that ethaboxam is effective in managing MR Pythium. Ethaboxam in combination with metalaxyl is commonly applied as seed treatments in commercial chickpea production.

Outcome of sexual reproduction in the Phytophthora infestans population in Estonian potato fields

In this study, the Estonian population of Phytophthora infestans was characterized with mating type, sensitivity to metalaxyl, virulence on 11 potato R-gene differentials and 12 SSR markers to show the outcome of potential sexual reproduction in the population. During the three years 2010–2012, 141 P. infestans isolates, collected from 23 potato fields, showed quite a high and stable frequency of the A2 mating type, 48% of the total population. In 87% of all sampled potato fields, both mating types were recorded, suggesting continuous sexual reproduction of P. infestans and possible oospore production. Metalaxyl-sensitive isolates prevailed in all three years (68 out of 99 isolates). Amongst the 95 isolates tested, 51 virulence races were found. The race structure was diverse, and most pathotypes were unique, appearing only once; the two most common pathotypes, 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11, comprised 35% of the population. The P. infestans population was genetically highly diverse and most of the multilocus genotypes (MLGs) appeared only once. Furthermore, all of the MLGs appeared in only one of the three sampling years. Our results confirm that the high diversity in the Estonian P. infestans population is most likely the result of frequent sexual reproduction, which benefits the survival, adaptability and diversity of the pathogen in the climate of North-Eastern Europe.

Fungitoxicity and synergism of mixtures of fluopicolide and pyraclostrobin against Phytophthora infestans

Fluopicolide and pyraclostrobin were new systemic fungicides with highly inhibitory activity on a broad spectrum of oomycetes, but so far the fungitoxicity of their mixtures on the different developmental stages of Phytophthora infestans and whether synergism exists have not been investigated. The joint-toxicity of the mixtures of fluopicolide with pyraclostrobin was determined against the different developmental stages (mycelial growth, zoospore release, cystospore germination and sporangial germination) of P. infestans and late blight on the leaf discs and the potted plants of potato, and whether the synergistic interaction exists in the mixtures of the two fungicides were evaluated in vitro and in vivo based on the synergistic ratios calculated with the Wadley formulas. The protective activity and curative activity against P. infestans and the efficacy duration in controlling potato late blight of the synergistic mixtures of the two fungicides were examined in this study. The results showed that mixtures of fluopicolide (F) and pyraclostrobin (P) at the ratios of 10:1 and 1:4 (F:P) exhibited synergistic interactions and had excellent inhibitory activity against almost all developmental stages of P. infestans. Synergistic interactions of the mixtures at the ratios of 1:4 and 10:1 (F:P) against the mycelial growth of metalaxyl-resistant isolates were more obvious than against the mycelial growth of metalaxyl-sensitive isolates. Synergistic interactions of the mixtures at the ratio of 1:4 (F:P) against the mycelial growth of the most of the tested isolates were more obvious than those of the mixtures at the ratio of 10:1 (F:P). The highest activity of the mixtures was observed against zoospore release at the ratio of 1:4 (F:P) with EC50 value of 0.0044 μg ml−1. The strongest synergistic interaction of the mixtures was observed against zoospore release at the ratio of 10:1 (F:P) with the synergistic ratio of 5.27. The mixtures of fluopicolide and pyraclostrobin at the ratios of 1:4 and 10:1 (F:P) showed synergistic interactions against P. infestans on the leaf discs and intact plants of potato. The EC50 values for protective activity and curative activity of the mixture at the ratio of 10:1 (F:P) were 0.63 μg ml−1 and 0.87 μg ml−1, and EC50 values of protective activity and curative activity of the mixture at the ratio of 1:4 (F:P) were 0.27 μg ml−1 and 0.44 μg ml−1, respectively. At 14 days after spraying of fungicide solutions, the disease control efficacy of the mixtures at the ratios of 10:1 and 1:4 (F:P) was still over 80% at the dosages of 50 μg ml−1 and 100 μg ml−1, and the disease control efficacy of the two mixtures remained at about 97% at the dosages of 200 μg ml−1, indicating long-lasting effective control efficacy of the two fungicides against potato late blight.

우리나라 골프장 잔디에서 분리한 Metalaxyl 저항성 Pythium spp.의 발생

Of 125 isolates collected from 35 golf courses, sensitivity of 44 isolates of Pythium species to metalaxyl was determined on corn meal agar with various concentrations of metalaxyl (0.1, 1.0, 10.0, 50.0, 100.0, and ). The isolates were able to be categorized into the sensitive and resistant groups based on hyphal growth measured in terms of colony diameters on the medium with . When compared with hyphal growth on the medium without metalaxyl, hyphal growth of the sensitive group which included 31 isolates was inhibited by on the medium with , whereas that of the resistant group which included 13 isolates grew well and the hyphal growth was inhibited only by . When of metalaxyl was included in the medium, hyphal growth of the sensitive and resistant groups was inhibited by , respectively. Occurrence of metalaxyl-resistant isolates of Pythium spp. not only from turfgrasses on golf courses but also from other crops was observed for the first time in Korea. Metalaxyl-resistant isolates occurred most frequently in P. graminicola. Control effects of metalaxyl were determined by applying metalaxyl after and before inoculation of 4 and 3 isolates of sensitive and resistant isolates of P. graminicola, respectively, to creeping bentgrass in pots. The minimum concentration of metalaxyl to control metalaxyl-sensitive isolates was , whereas the disease caused by the metalaxyl-resistant isolates could not be controlled with of metalaxyl. The disease was controlled more effectively by an application of metalaxyl prior to inoculation than after occurrence of the disease.

Resistance to Mefenoxam and Metalaxyl Among Field Isolates of Phytophthora capsici Causing Phytophthora Blight of Bell Pepper.

Incidence of Phytophthora blight in bell pepper fields that were sprayed for the first time with Ridomil Gold (mefenoxam) according to labeled recommendations was higher in North Carolina in 1997 than in previous years. Mefenoxam is the more active enantiomer contained in the racemic fungicide metalaxyl. A total of 150 isolates were obtained from 17 fields at eight grower locations. Among isolates from all locations, 30% were classified as sensitive, 10% as intermediate, and 59% were resistant to mefenoxam. Mefenoxam-resistant isolates were found in 82% of the fields sampled (14 of 17 fields). The proportion of resistant isolates in individual (fields ranged from 28 to 100%. The mean effective concentration (EC50) values for mefenoxam-sensitive isolates was 0.568 μg ml-1 (ranging from 0.12 to 1.1 μg ml-1), whereas the mean EC50 value for mefenoxam-resistant isolates was 366.5 μg ml-1 (ranging from 3 to 863 μg ml-1). The mean EC50 value for metalaxyl-sensitive isolates was 0.27 μg ml-1 (ranging from 0.00002 to 1.3 μg ml-1) and for metalaxyl-resistant isolates was 470.34 μg ml-1 (ranging from 10 to 966 μg ml-1). The greatest proportion of resistant isolates came from fields where mefenoxam was used alone rather than in combination with other fungicides. Both mating types were found among resistant isolates, suggesting that these isolates may persist in soil in subsequent years. Field isolates of Phytophthora capsici resistant to mefenoxam on pepper have not been reported previously and now pose new challenges for management of this important disease.

Commercial Fungicide Formulations Induce In Vitro Oospore Formation and Phenotypic Change in Mating Type in Phytophthora infestans

ABSTRACT A wide range of commercially formulated fungicides cause in vitro effects on mating behavior in specific isolates of Phytophthora infestans, the causal agent of late blight of potato and tomato. Four isolates of P. infestans representing each of the four common US genotypes, US-1, US-6, US-7, and US-8 and varying in their sensitivity to metalaxyl, were exposed to a variety of fungicides used to control late blight in petri dish assays at concentrations ranging from 1 to 100 mug a.i./ml. Exposure of each of these normally heterothallic single mating type isolates of P. infestans to 9 of the 11 commercial fungicide formulations tested resulted in the formation of oospores after 2 to 4 weeks. The highest numbers of oospores were formed on media amended with Ridomil 2E (metalaxyl) and Ridomil Gold EC (mefenoxam) at 0.1 to 10 mug a.i./ml, averaging as many as 471 and 450 oospores per petri dish, respectively. Several other fungicides including Maneb, Manzate (Mancozeb), Curzate (cymoxanil + mancozeb), and Acrobat MZ (dimethomorph + mancozeb) also induced oospore formation, producing from 0 to 200 oospores per plate at fungicide concentrations from 0.1 to 10 mug a.i./ml. The metalaxyl resistant isolates formed oospores in response to the fungicides more often than the metalaxyl sensitive isolates. No oospores were formed on media amended with Bravo (chlorothalonil) or Tattoo C (chlorothalonil + propamocarb HCl) and these compounds completely suppressed growth of the isolates at 0.1 and 1 mug a.i./ml. Three metalaxyl resistant A2 isolates mated with both A1 and A2 isolates after exposure to the fungicides Ridomil 2E and Ridomil Gold EC. Alterations in mating type expression were also observed in a metalaxyl sensitive A1 isolate after exposure to Benlate (benomyl). Copious amounts of chemicals are applied annually to potato and tomato production areas to control late blight. Our results indicate that a wide range of chemically diverse fungicides can induce normally heterothallic metalaxyl resistant isolates of P. infestans to form oospores in vitro after short exposures to the fungicides.

Toxicity of propamocarb to the late blight fungus on potato

Propamocarb (Previcur-N; propyl-[3-dimethylamino-propyl] carbamate-monohydrochloride) was testedin vivo against 32 field isolates ofPhytophthora infestans from six countries. Fungicide dosages required to achieve 90% control of the blight ranged between 676 and 1530 ppm a.i. in potted potato (cv. ‘Alpha’) plants and between 1135 and 2648 ppm in potato tuber slices. Isolates from Israel were less sensitive to the fungicide than isolates from Europe or North America. Toxicity of propamocarb was not related to resistance or sensitivity to phenylamide fungicides (e.g. metalaxyl). Nevertheless, most metalaxyl-resistant isolates from Israel were less sensitive to propamocarb than most metalaxyl-sensitive isolates from this country. Monocyclic epidemics conducted with the 20 Israeli isolates in the field showed that 1081–2012 ppm of the fungicide was required to achieve 90% control of the disease. Laboratory experiments revealed that the fungicide was poorly active against sporangial germination and had a limited curative efficacy. It exhibited a translaminar translocation in leaves but a poor acropetal or basipetal systemicity from foliage. Propamocarb + mancozeb mixtures (1:1, v/w) were synergistically effective in controlling the blight. Growers in Israel use tank mixtures of propamocarb (Dynone) and mancozeb to combat late blight in potato fields where phenylamide-resistant isolates ofP. infestans are prevalent.