California Pepper Commission 2015 RESEARCH PROJECT PROPOSAL
Research Project Proposal
California Pepper Commission
531-D North Ata Avenue, Dinuba, CA 93618-3203
Monitoring of azole and strobilurin fungicide resistance in pepper powdery mildew
Ioannis Stergiopoulos, Assistant Professor Department of Plant Pathology, University of California Davis 578 Hutchison Hall, One Shields Avenue, Davis, CA 95616-8751, USA Tel: +1 (530) 400-9802; Fax: +1 (530) 752-5674
Steven T. Koike, Cooperative Extension Farm Advisor, Monterey County (collaborator Joe Nunez, Cooperative Extension Farm Advisor, Kern County (collaborator) Tom Turini, Cooperative Extension Farm Advisor, Fresno County (collaborator Brenna Aegerter, Cooperative Extension Farm Advisor, San Joaquin County (collaborator) Gene Miyao, Cooperative Extension Farm Advisor, Yolo County (collaborator)
Start and End Date:
April 1st 2015 - March 31st 2016
$20,000 will be requested for this project
1.1 Rationale: Powdery mildews are obligate biotrophic fungi that cause extensive diseases in crops. In pepper, powdery mildew is caused by the species Leveillula taurica, which commonly infects the leaves, thus causing defoliation and reducing the photosynthetic capacity of the plants. The fungus first appeared in CA in the early 1990's and since then has been a recurring problem, mainly on chilli and bell peppers. In addition to peppers, in California the fungus is also known to cause extensive damage in field-grown tomatoes. Powdery mildew epidemics occur almost yearly in most pepper and tomato growing areas of California and can significantly impact fruit production and quality. In this respect, a direct correlation between percentage of mildew infection and yield loss is said to exist. In North America, average yield losses to powdery mildew are estimated to 10-15%, although losses of 50-60% have occasionally been reported for peppers in California as well. Current control methods for powdery mildew mostly rely on the use of chemical control agents, such as commercial fungicides and sulfur- dusts. Azole and strobilurin fungicides, in particular, are the two fungicide classes that have been used extensively in the field as an alternative or in addition to sulfur treatments. Rally® (myclobutanil, an azole fungicide), CabrioTM (pyraclostrobin, a strobilurin fungicide), QuadrisTM (azoxystrobin, a strobilurin fungicide), and Quadris TopTM (azoxystrobin and difenoconazole, a mixture of a strobilurin and an azole fungicide), are perhaps the most widely used compounds in California against powdery mildew in peppers and/or tomato. The continuous use, however, of these fungicides in the fields increases the danger for fungicide resistance development and cases of possible shifts in the sensitivity of the fungus towards fungicides like Rally and Cabrio have already been reported.
1.2 Results from on-going research on fungicide resistance of L. taurica on tomato: Prompted by reports from tomato growers for sporadic failure of mildew control in the field, in 2014 we investigated whether the continuous use of azole and strobilurin fungicides has sparked the appearance of resistant strains of L. taurica in California. Since resistance development in fungi is frequently correlated with mutations at the target site of the fungicides, to achieve our goal we followed an approach of first cloning and subsequently screening L. taurica populations collected from infected tomato plants for mutations in the CYP51 and the mitochondrial cytochrome b (cytb) genes that encode for the enzymatic targets of azole and strobilurin fungicides, respectively. Analysis of cytb showed that strains with mutations conferring resistance to strobilurins, such as the notorious G143A mutation, are already widely present in tomato fields. However, the analysis also indicated that the presence of this mutation in field strains of L. taurica is associated with mitochondrial heteroplasmy for the cytb gene, suggesting that resistance in the fields is not yet complete but rather correlates with the portion of mitochondria in each strain carrying the mutated cytb allele. Thus, it is imperative at this point to closely monitor the situation and establish whether the continuous use of QoIs would lead to an increase in the proportion of mitochondria carrying the G143A mutation in cytb. Along the same lines, it is also important to examine how stable the observed heteroplasmy is in the absence of QoI fungicides and whether heteroplasmic isolates for the G143A mutation could be reverted back to wild-type. This might be possible since the presence of both the wild-type and mutated mitochondria in a single strain could imply that there is a fitness penalty associated with the G143A mutation. If such is the case, then extending the fungicide- free cultivation period for a number of fungal generations that can be defined experimentally, could potentially reverse fungicide resistance and reset the situation in the fields. Currently we are also investigating whether mutations are present in CYP51 gene as well.
1.3 Specific objectives for pepper: The presence of mutations for strobilurin resistance (and perhaps for azoles as well) in a large portion of the pathogen population that is found on tomatoes is an alarming indication that we are faced with the development of fungicide resistance, and thus there is a need to monitor the situation. Currently, it is unknown whether strains of L. taurica infecting tomato can also readily jump to pepper and vise-versa or whether there are subpopulations of the pathogen specializingon one or the other host. Whichever the case, the presence of L. taurica strains on tomato with a spectrum of sensitivities against strobilurin fungicides implies that a similar situation might exist for strains infecting pepper as well. Since we find mixed populations of fungicide resistant and sensitive strains, it implies that resistance in not yet complete, and thus action needs to be taken to prevent its further development. Notably, for pathogens such as the wheat pathogen Septoria trittici, resistance development against strobilurin fungicides took place within the span of just a couple of years since the first appearance of resistant strains in the field. Based on our findings regarding the presence in California of L. taurica strains infecting tomato with resistant against fungicides, our objectives for pepper are:
Objective 1:To investigate whether the continuous use of azole and strobilurin fungicides has sparked the appearance of resistant strains of L. taurica on pepper.
Objective 2: To determine the distribution across the State of California of potentially fungicide resistant strains of L. taurica collected from pepper, and examine the stability of resistance within and between growing seasons.
The results from this study will help design a strategy for the successful management of fungicide resistance of powdery mildew in California that will help preserve yields, improve quality of products, and prolong the commercial life of agrochemicals. Moreover, it could potentially reduce production costs and the impact of fungicides on the environment and residents' health, by maximizing the efficacy of applied chemicals. Results obtained from this proposal will also be used as a reference point for establishing a surveillance system for early detection of shifts in sensitivity to currently used fungicides. Such information is critical for preventing future epidemics of the pathogen and the breakdown of control in the field. Finally, the data will be ultimately used for the development of rapid and reliable molecular-based genotyping techniques such as PCR-based methods, suitable for routine high- throughput monitoring of fungicide resistance in field populations of the pathogen.
To monitor for shifts in the frequency of fungicide resistant strains collected from pepper, we will follow an approach similar to the one that we have followed for tomato, by screening field populations of the pathogen for mutations in the CYP51 and cytb genes that confer resistance to azoles and strobilurin fungicides, respectively. Sampling will be performed every second week throughout the growing season from the major California counties where peppers are mostly grown and will include both field and greenhouse-grown peppers. All relevant information, such as location, date, pepper variety, fungicide- spray history (if known), will be meticulously recorded. Based on our experience with tomato, we anticipate that we will be able to analyze ~150 strains collected from pepper plants throughout the growing season. Since we have already cloned in 2014 the CYP51 and cytb genes from L. taurica and developed species-specific primers for PCR amplifications, monitoring for mutations in these two genes becomes very straightforward and cost effective. Briefly, leaves showing symptoms or signs of powdery mildew will be collected from several pepper plants and locations across the state. The infected material is then brought to the lab and DNA extractions are performed from individual leaves. Using gene-specific primers the CYP51 and cytb genes are then amplified from these DNA samples, sequenced, and the produced electropherograms are analyzed for the presence of mutations. In this way we can maximize the number of strains that are analyzed for mutations, without the burden of generating monospore cultures first.
3. SUGGESTED BUDGET DETAIL AND BUDGET JUSTIFICATION
3.1 Costs Breakdown:
PI salary (1 month summer compensation, 40%): $3,156
PI benefits: $537
Graduate student salary (Fall quarter, 100%): $6,881
Graduate student benefits: $5,603
Sequencing costs and lab Supplies (UC Davis site): $3,823
3.2 Personnel: 40% of one month summer compensation ($3,156) is requested for Assistant Professor Ioannis Stergiopoulos. Dr Stergiopoulos is the PI on the proposal and is overseeing the entire project, managing and directing all efforts, including its design, implementation, analysis of the results, and potential trouble-shooting. Dr Stergiopoulos is also actively working in lab for this project performing DNA extractions, PCR amplifications, and analysis of all the sequencing results. The fridge benefit rate (40%) for the PI is $537.
Support for one quarter (Fall quarter: $6,881, 100% appointment) is also requested for a graduate student who will work in the lab doing DNA extractions, PCR amplifications and cloning of PCR products to cloning vectors. The fridge benefit rate (100%)for the graduate student is $5,603.
Supplies: $3,823 is requested for supplies, including the purchasing of DNA isolation kits, PCR reagents, Primers, and sequencing of the PCR fragments. Sequencing costs are calculated to $5 per reaction (2 genes X 150 samples X $5 = $1,500). A Qiagen DNeasy Plant mini kit for 250 reactions costs
$903. Supertaq Polymerases (per 600 rxns) costs $480. Other costs, like PCR tubes, pipette tips, pGemt kit, etc are calculated to ~$940.
4. APPROVAL SIGNATURES
Project has been accepted by the UC Davis Sponsored Programs Office (SPO Project Number 201502308)
Dr Ioannis Stergiopoulos