Martin I. Chilvers

A real-time, quantitative PCR seed assay for Botrytis spp. that cause neck rot of onion

A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

Didymella pisi sp. nov., the teleomorph of Ascochyta pisi

The anamorphic pycnidial fungus Ascochyta pisi is one member of a species complex that causes Ascochyta blight of pea, a potentially devastating disease. The teleomorphic state of this fungus was induced under laboratory conditions. Using morphological and molecular characters, we placed the teleomorph within the genus Didymella as D. pisi and describe a heterothallic mating system using a PCR-based mating type assay and in vitro crosses. We compare D. pisi with other Didymella spp. with which it might be confused.

Biology, Yield loss and Control of Sclerotinia Stem Rot of Soybean

Sclerotinia stem rot (also known as white mold) of soybean is a significant yield-limiting problem in the North Central production region. This disease, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary, varies in incidence and severity from year to year because of its sensitivity to weather conditions. Losses because of Sclerotinia stem rot can be substantial when environmental conditions and management practices favor high yield potential. Employing a disease management plan based on knowledge of field history and best disease management practices can help reduce losses from Sclerotinia stem rot. An effective disease management plan integrates several management tactics that include cultural practices, varietal resistance, as well as chemical and biological control. Understanding how different environmental variables and management practices influence infection by S . sclerotiorum and disease development are important to optimize disease management and reduce losses. This profile summarizes research-based knowledge of Sclerotinia stem rot, including the disease cycle, the scope of the losses that can occur because of this disease, how to identify both the pathogen S . sclerotiorum and the disease, and current management recommendations.

Ecological genetic divergence of the fungal pathogen Didymella rabiei on sympatric wild and domesticated Cicer spp. (Chickpea).

ABSTRACT For millennia, chickpea (Cicer arietinum) has been grown in the Levant sympatrically with wild Cicer species. Chickpea is traditionally spring-sown, while its wild relatives germinate in the autumn and develop in the winter. It has been hypothesized that the human-directed shift of domesticated chickpea to summer production was an attempt to escape the devastating Ascochyta disease caused by Didymella rabiei. We estimated genetic divergence between D. rabiei isolates sampled from wild Cicer judaicum and domesticated C. arietinum and the potential role of temperature adaptation in this divergence. Neutral genetic markers showed strong differentiation between pathogen samples from the two hosts. Isolates from domesticated chickpea demonstrated increased adaptation to higher temperatures when grown in vitro compared with isolates from the wild host. The distribution of temperature responses among progeny from crosses of isolates from C. judaicum with isolates from C. arietinum was continuous, suggesting polygenic control of this trait. In vivo inoculations of host plants indicated that pathogenic fitness of the native isolates was higher than that of their hybrid progeny. The results indicate that there is a potential for adaptation to higher temperatures; however, the chances for formation of hybrids which are capable of parasitizing both hosts over a broad temperature range are low. We hypothesize that this pathogenic fitness cost is due to breakdown of coadapted gene complexes controlling pathogenic fitness on each host and may be responsible for maintenance of genetic differentiation between the pathogen demes.

MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network

Large-scale high-throughput plant phenotyping (sometimes called phenomics) is becoming increasingly important in plant biology and agriculture and is essential to cutting-edge plant breeding and management approaches needed to meet the food and fuel needs for the next century. Currently, the application of these approaches is severely limited by the availability of appropriate instrumentation and by the ability to communicate experimental protocols, results and analyses. To address these issues, we have developed a low-cost, yet sophisticated open-source scientific instrument designed to enable communities of researchers, plant breeders, educators, farmers and citizen scientists to collect high-quality field data on a large scale. The MultispeQ provides measurements in the field or laboratory of both, environmental conditions (light intensity and quality, temperature, humidity, CO2 levels, time and location) and useful plant phenotypes, including photosynthetic parameters—photosystem II quantum yield (ΦII), non-photochemical exciton quenching (NPQ), photosystem II photoinhibition, light-driven proton translocation and thylakoid proton motive force, regulation of the chloroplast ATP synthase and potentially many others—and leaf chlorophyll and other pigments. Plant phenotype data are transmitted from the MultispeQ to mobile devices, laptops or desktop computers together with key metadata that gets saved to the PhotosynQ platform (https://photosynq.org) and provides a suite of web-based tools for sharing, visualization, filtering, dissemination and analyses. We present validation experiments, comparing MultispeQ results with established platforms, and show that it can be usefully deployed in both laboratory and field settings. We present evidence that MultispeQ can be used by communities of researchers to rapidly measure, store and analyse multiple environmental and plant properties, allowing for deeper understanding of the complex interactions between plants and their environment.

Corn yield loss estimates due to diseases in the United States and Ontario, Canada from 2012 to 2015.

Annual decreases in corn yield caused by diseases were estimated by surveying members of the Corn Disease Working Group in 22 corn-producing states in the United States and in Ontario, Canada, from 2012 through 2015. Estimated loss from each disease varied greatly by state and year. In general, foliar diseases such as northern corn leaf blight, gray leaf spot, and Goss’s wilt commonly caused the largest estimated yield loss in the northern United States and Ontario during nondrought years. Fusarium stalk rot and plant-parasitic nematodes caused the most estimated loss in the southernmost United States. The estimated mean economic loss due to yield loss by corn diseases in the United States and Ontario from 2012 to 2015 was

A Coordinated Effort to Manage Soybean Rust in North America: A Success Story in Soybean Disease Monitoring

76.51 USD per acre. The cost of disease-mitigating strategies is another potential source of profit loss. Results from this survey will provide scientists, breeders, government, and educators with data to help inform and prioritize research, policy, and educational efforts in corn pathology and disease management. M U E L L E R E T A L . , P L A N T H E A L T H P R O G R E S S 1 7 (2 0 1 6 )

Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei

Existing crop monitoring programs determine the incidence and distribution of plant diseases and pathogens and assess the damage caused within a crop production region. These programs have traditionally used observed or predicted disease and pathogen data and environmental information to prescribe management practices that minimize crop loss. Monitoring programs are especially important for crops with broad geographic distribution or for diseases that can cause rapid and great economic losses. Successful monitoring programs have been developed for several plant diseases, including downy mildew of cucurbits, Fusarium head blight of wheat, potato late blight, and rusts of cereal crops. A recent example of a successful disease-monitoring program for an economically important crop is the soybean rust (SBR) monitoring effort within North America. SBR, caused by the fungus Phakopsora pachyrhizi, was first identified in the continental United States in November 2004. SBR causes moderate to severe yield losses globally. The fungus produces foliar lesions on soybean (Glycine max) and other legume hosts. P. pachyrhizi diverts nutrients from the host to its own growth and reproduction. The lesions also reduce photosynthetic area. Uredinia rupture the host epidermis and diminish stomatal regulation of transpiration to cause tissue desiccation and premature defoliation. Severe soybean yield losses can occur if plants defoliate during the mid-reproductive growth stages. The rapid response to the threat of SBR in North America resulted in an unprecedented amount of information dissemination and the development of a real-time, publicly available monitoring and prediction system known as the Soybean Rust-Pest Information Platform for Extension and Education (SBR-PIPE). The objectives of this article are (i) to highlight the successful response effort to SBR in North America, and (ii) to introduce researchers to the quantity and type of data generated by SBR-PIPE. Data from this system may now be used to answer questions about the biology, ecology, and epidemiology of an important pathogen and disease of soybean.

Oomycete Species Associated with Soybean Seedlings in North America—Part I: Identification and Pathogenicity Characterization

Ascochyta rabiei produces and accumulates one of the well-known fungal polyketides, 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin), in asexual and sexual fruiting bodies. Degenerate PCR primers were used to isolate an ArPKS1 of A. rabiei encoding a polypeptide with high similarity to polyketide synthase (PKS) involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants but caused no significant reduction of pathogenicity to chickpea. Pycnidiospores in ArPKS1-mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. This study demonstrated that A. rabiei uses a DHN-melanin pathway for pigmentation of pycnidia and this molecule may protect pycnidiospores from UV irradiation.

Rapid transcriptome characterization and parsing of sequences in a non-model host-pathogen interaction; pea- Sclerotinia sclerotiorum

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.