Margaret T. McGrath

Fungicide Resistance in Cucurbit Powdery Mildew: Experiences and Challenges

Fungicides are an important tool for managing cucurbit powdery mildew, which is a major production problem in many areas of the world (89). Application of fungicides is presently the principal practice in most cucurbit crops for managing powdery mildew. Fungicides that are systemic or have translaminar activity are needed to obtain adequate protection of abaxial leaf surfaces, where conditions are more favorable for development of the pathogen than on adaxial surfaces (Figs. 1 and 2) (80). Unfortunately, these fungicides generally have a high risk of developing resistance because they have specific modes of action, and powdery mildew fungi have a high potential for resistance development. This has been especially true for the predominate cucurbit powdery mildew fungus, Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff (also known as Sphaerotheca fusca (Fr.) S. Blumer and S. fuliginea (Schlechtend.:Fr.) Pollacci). Fungicide resistance is the stable, inheritable adjustment by a fungus to a fungicide, resulting in reduced sensitivity of the fungus to the fungicide. This ability is obtained through evolutionary processes. Systemic and translaminar fungicides are generally more at-risk for resistance development than contact fungicides because they typically have specific, single-site mode of action, which means they are active against only one point in one metabolic pathway in a pathogen. When resistance results from modification of a single major gene, pathogens are either resistant or sensitive to the pesticide, and disruptive selection occurs. Resistance in this case is seen as complete loss of disease control that cannot be regained by using higher rates or more frequent fungicide applications. This type of resistance is commonly referred to as “qualitative resistance” and is exemplified by resistance to benzimidazole fungicides, which results from a conformational change at the target site in various pathogens. When resistance results from modification of several interacting genes, pathogens exhibit a range in sensitivity to the fungicide depending on the number of gene changes. Variation in sensitivity within the population is continuous or unimodal, and selection occurs in a directional manner. Resistance in this case is seen as an erosion of disease control that can be regained by using higher rates or more frequent applications. Additional selection in the pathogen may eventually result in complete loss of control. This type is commonly referred to as “quantitative resistance” and is exemplified by resistance to demethylation inhibiting (DMI) fungicides. Several mechanisms of DMI resistance seem to be operating in plant pathogens based on the broad range of phenotypic sensitivities observed (32,48). Possible mechanisms, identified mainly through work with model fungal systems, include decreased accumulation of fungicide due to increased efflux or decreased uptake, deficiency in the target site, reduced affinity for the target site, tolerance of toxic sterols, detoxifi-

The 2009 late blight pandemic in the eastern United States - causes and results

The tomato late blight pandemic of 2009 made late blight into a household term in much of the eastern United States. Many home gardeners and many organic producers lost most if not all of their tomato crop, and their experiences were reported in the mainstream press. Some CSAs (Community Supported Agriculture) could not provide tomatoes to their members. In response, many questions emerged: How did it happen? What was unusual about this event compared to previous late blight epidemics? What is the current situation in 2012 and what can be done? Its easiest to answer these questions, and to understand the recent epidemics of late blight, if one knows a bit of the history of the disease and the biology of the causal agent, Phytophthora infestans.

Increased resistance to triadimefon and to benomyl in Sphaerotheca fuliginea populations following fungicide usage over one season

Fungicide sensitivity of Sphaerotheca fuliginea was monitored during powdery mildew epidemics in research plots and production fields of pumpkin. Benomyl-resistant isolates (insensitive to 200 μg/ml) were detected in all populations before benomyl was used. The only triadimefon-resistant isolate (insensitive to 100 μg/ml) detected prior to treatment was near triadimefon-treated plants. Populations in nontreated fields did not change appreciably. Fungicide application had a large impact on fungicide resistance. Proportion of resistant isolates in research plots treated four times with triadimefon plus chlorothalonil shifted from 0 to 81% for triadimefon and from 30 to 69% for benomyl between 15 August and 19 September 1991; proportion shifted from 3 to 100% for triadimefon and from 10 to 44% for benomyl between 14 August and 17 September 1992. The shift occurred rapidly: proportion of resistant isolates shifted within 2 weeks from 0 to 96% for triadimefon and from 10 to 74% for benomyl following two applications of triadimefon in a commercial field in August 1992. Most isolates (87%) from fungicide-treated fields tolerated triadimefon at 100 or 200 μg/ml. These were considered triadimefon-resistant because fungicide performance declined during September. However, control was commercially acceptable. Of 122 isolates collected from fungicide-treated pumpkin, one was benomyl-resistant, triadimefon-sensitive; 20 were benomyl-sensitive, triadimefon-resistant; six were sensitive to both fungicides; and 95 were resistant to both with 77 of these insensitive to triadimefon at 200 μg/ml. In contrast, 57 of 92 isolates from nontreated populations were sensitive to both fungicides, none were resistant to both, and 32 were resistant to benomyl.

Population structure and resistance to mefenoxam of Phytophthora capsici in New York State

In 2006, 2007, and 2008, we sampled 257 isolates of Phytophthora capsici from vegetables at 22 sites in four regions of New York, to determine variation in mefenoxam resistance and population genetic structure. Isolates were assayed for mefenoxam resistance and genotyped for mating type and five microsatellite loci. We found mefenoxam-resistant isolates at a high frequency in the Capital District and Long Island, but none were found in western New York or central New York. Both A1 and A2 mating types were found at 12 of the 22 sites, and we detected 126 distinct multilocus genotypes, only nine of which were found at more than one site. Significant differentiation (FST) was found in more than 98% of the pairwise comparisons between sites; approximately 24 and 16% of the variation in the population was attributed to differences among regions and sites, respectively. These results indicate that P. capsici in New York is highly diverse, but gene flow among regions and fields is restricted. Therefore, each field needs to be considered an independent population, and efforts to prevent movement of inoculum among fields need to be further emphasized to prevent the spread of this pathogen.

Emergence of Groundnut ringspot virus and Tomato chlorotic spot virus in Vegetables in Florida and the Southeastern United States

Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) are two emerging tospoviruses in Florida. In a survey of the southeastern United States, GRSV and TCSV were frequently detected in solanaceous crops and weeds with tospovirus-like symptoms in south Florida, and occurred sympatrically with Tomato spotted wilt virus (TSWV) in tomato and pepper in south Florida. TSWV was the only tospovirus detected in other survey locations, with the exceptions of GRSV from tomato (Solanum lycopersicum) in South Carolina and New York, both of which are first reports. Impatiens (Impatiens walleriana) and lettuce (Lactuca sativa) were the only non-solanaceous GRSV and/or TCSV hosts identified in experimental host range studies. Little genetic diversity was observed in GRSV and TCSV sequences, likely due to the recent introductions of both viruses. All GRSV isolates characterized were reassortants with the TCSV M RNA. In laboratory transmission studies, Frankliniella schultzei was a more efficient vector of GRSV than F. occidentalis. TCSV was acquired more efficiently than GRSV by F. occidentalis but upon acquisition, transmission frequencies were similar. Further spread of GRSV and TCSV in the United States is possible and detection of mixed infections highlights the opportunity for additional reassortment of tospovirus genomic RNAs.

Resistance to Triadimefon and Benomyl: Dynamics and Impact on Managing Cucurbit Powdery Mildew

Frequency of fungicide-resistant strains of Podosphaera xanthii on pumpkins in New York before treatment varied from 3 to 80% for the demethylation inhibiting (DMI) fungicide triadimefon and from 0 to 48% for the benzimidazole fungicide benomyl between 1993 and 1996. When the initial frequency of triadimefon-resistant strains was less than 55%, one application of triadimefon plus chlorothalonil was effective. This application was made after reaching the action threshold of one leaf with powdery mildew symptoms per 50 old leaves (defined as the oldest third of the foliage). The frequency of triadimefon-resistant strains increased from 3 to 71% by 20 days after the first fungicide application in 1993. Triadimefon in the second application did not contribute to control. Loss of efficacy was due to resistance because, compared with triadimefon-treated pumpkins, pumpkins treated with other systemic fungicides were less severely infected by powdery mildew on abaxial leaf surfaces where the companion multi-site contact fungicide contributes little to control. Triadimefon was not effective in 1995 when 80% of the pathogen population was resistant before treatment. Benomyl was effective in 1995, but not in 1996 when 48% of the isolates tested were resistant to both benomyl and triadimefon before treatment. An in-field seedling assay was developed to determine local occurrence of resistant strains before the first treatment was needed. Although sensitivity of the pathogen population to the DMI fungicides myclobutanil and propiconazole also decreased after they were applied, these fungicides were more effective than triadimefon.

First Report of the Cucurbit Powdery Mildew Fungus (Podosphaera xanthii) Resistant to Strobilurin Fungicides in the United States

Resistance to strobilurin fungicides was documented in isolates collected from three fungicide efficacy experiments conducted in research fields in Georgia (GA), North Carolina (NC), and New York (NY). In these fields in 2002, strobilurins (fungicide group 11, quinone outside inhibitors [QoI]) when used alone on a 7-day schedule (use pattern not labeled) did not effectively control cucurbit powdery mildew. Strobilurin efficacy declined dramatically after the second application in New York (3). Efficacy also was reduced in commercial fields in Kentucky and research fields in Arizona, California, Kentucky, Illinois, Michigan, and Virginia in 2002 where strobilurins were used predominantly or exclusively. Isolates were collected on 22 July and 8 and 17 October after the last of four, five, and five applications of strobilurin (trifloxystrobin formulated as Flint or azoxystrobin formulated as Quadris) in experiments conducted by J. D. Moore in Chula, GA, M. McGrath in Riverhead, NY, and G. J. Holmes in Clayton, NC, respectively. A leaf-disk bioassay was used to determine fungicide sensitivity (2). Strobilurin sensitivity was determined using trifloxystrobin at 0, 0.5, 5, 50, and 100 μg/ml. Four of nine NY isolates, 19 of 21 GA isolates, and 13 of 15 NC isolates were resistant to strobilurins (grew well on disks treated with trifloxystrobin at 100 μg/ml). The geometric mean of the azoxystrobin baseline was 0.258 μg/ml for Podosphaera xanthii isolates collected in 1998 and 1999 in North America (4). Poor control with strobilurins under field conditions was associated with reduced sensitivity in vitro. Strobilurin sensitivity appeared to be qualitative as reported elsewhere (1). Two sensitive and three resistant isolates responded similarly when tested in another laboratory using kresoxim-methyl and pyraclostrobin (H. Ypema, personal communication). These findings and experiences elsewhere with QoI-resistant P. xanthii indicate that cross-resistance probably extends among multiple QoIs (1). Strobilurins have been available for commercial use in the United States since 1998, when azoxystrobin received Section 18 registration in some states. Federal registration was granted in March 1999. Strobilurin resistance was detected after 2 years of commercial use elsewhere in the world (1). All isolates tested in the current study were from research fields where selection pressure for resistance could have been higher than in commercial fields where strobilurins are used with demethylation inhibitors (DMIs; fungicide group 3) and contact fungicides in alternation or tank mixtures to prevent or delay resistance development. Resistance in commercial fields will reduce the utility of strobilurins, including those not yet registered, and eliminate an important tool for managing DMI resistance. Strobilurins and DMIs are the only systemic fungicides registered for cucurbit powdery mildew in the United States. Managing DMI resistance may be challenged by multiresistant strains. Strobilurin-resistant isolates also exhibited reduced sensitivity to DMIs, tolerating triadimefon at 50 to 100 μg/ml (2). One suggestion to improve resistance management is to apply a contact fungicide with strobilurins as well as DMIs. References: (1) H. Ishii et al. Phytopathology 91:1166, 2001. (2) M. T. McGrath et al. Plant Dis. 80:697, 1996. (3) M. T. McGrath and N. Shishkoff. Fungic. Nematic. Tests. (In press). (4) G. Olaya et al. Phytopathology (Abstr.) 90 (suppl):S57, 2000.

Basil Downy Mildew (Peronospora belbahrii): Discoveries and Challenges Relative to Its Control

Basil (Ocimum spp.) is one of the most economically important and widely grown herbs in the world. Basil downy mildew, caused by Peronospora belbahrii, has become an important disease in sweet basil (O. basilicum) production worldwide in the past decade. Global sweet basil production is at significant risk to basil downy mildew because of the lack of genetic resistance and the ability of the pathogen to be distributed on infested seed. Controlling the disease is challenging and consequently many crops have been lost. In the past few years, plant breeding efforts have been made to identify germplasm that can be used to introduce downy mildew resistance genes into commercial sweet basils while ensuring that resistant plants have the correct phenotype, aroma, and tastes needed for market acceptability. Fungicide efficacy studies have been conducted to evaluate current and newly developed conventional and organic fungicides for its management with limited success. This review explores the current efforts and progress being made in understanding basil downy mildew and its control.

Survival and spread of Phytophthora capsici on Long Island, New York

Phytophthora capsici is an oomycete soil-borne plant pathogen that causes root, fruit and foliar disease on a variety of vegetables. The epidemiology and population structure varies depending on the region surveyed and our objective was to investigate survival and spread on farms on Long Island, New York using single nucleotide polymorphism (SNP) markers. A total of 373 P. capsici isolates were collected from pumpkin, pepper, watermelon and snap bean on 15 farms. Both mating type were recovered from most locations. Genotypic analysis was conducted using 14 SNP loci located primarily within genes. A total of 128 unique multi-locus genotypes were identified. Of these, 54 appear to be clonal lineages ranging in size from 2 to 26 members. Most clonal lineages were recovered during the same year. Our results indicate that both sexual and clonal reproduction play important roles in the epidemiology of P. capsici on Long Island, NY, USA. The implications for managing the disease are discussed.

Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis

Abstract Real-time PCR assays using locked nucleic acid (LNA) probes and high resolution melt (HRM) analysis were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively. The assays were based on a previously identified single nucleotide polymorphism (SNP) in the cytochrome oxidase subunit II (cox2) gene that differentiates the two species. Sequencing of the same region from 15 P. cubensis isolates collected in New York State for the current study confirmed that all isolates shared the conserved SNP. LNA probes were specific and sensitive, detecting as few as 10 sporangia for both species and as little as 1 fg P. cubensis total DNA and 10 fg P. humuli total DNA. The LNA assay detected both pathogens from air sampled using spore traps placed in vegetable fields and a hop yard during the summers of 2013 and 2014 and correctly diagnosed symptomatic leaf tissue. High resolution melt analysis (HRM) correctly identified all tested isolates as well as those isolates from symptomatic plants collected in the field. The LNA and HRM assays correctly identified both organisms when tested independently in a second laboratory. The results confirm that the LNA and HRM assays developed can provide reliable identification of both species despite the high molecular similarity of the cox2 gene.