Biocontrol of Stemphylium vesicarium and Pleospora allii on Pear by Bacillus subtilis and Trichoderma spp.: Preventative and Curative Effects on Inoculum Production

Trials under controlled and field conditions were conducted to establish the effect of strategies of application of biological control agents (BCAs) in the reduction of Stemphylium vesicarium and Pleospora allii inoculum production on pear leaf debris. Six BCAs based on different strains of Trichoderma spp. (Tr1, Tr2) and Bacillus subtilis (Bs1, Bs2, Bs3 and Bs4) were evaluated. Two strategies were tested in controlled experiments: application before (preventative strategy) or after (curative strategy) pear leaf debris colonization by S. vesicarium, evaluating the growth inhibition and sporulation of S. vesicarium and the pseudothecia production of P. allii. When the BCAs were applied preventatively, the efficacy of treatments based on B. subtilis was higher than those based on Trichoderma spp. in controlling the pathogen colonization, but that of controlling the inoculum production of S. vesicarium and P. allii was similar. However, when the BCAs were applied curatively, Trichoderma based products were more effective. In field trials, Trichoderma spp. Tr1 and B. subtlilis Bs1 produced a consistent 45–50% decrease in the number of S. vesicarium conidia trapped compared to the non-treated control. We conclude that Bacillus subtilis Bs1 and Trichoderma spp. Tr1 and Tr2 can be expected to reduce fungal inoculum during the pear vegetative period by at least 45–50%. Additionally, Trichoderma spp. Tr1 and Tr2 have the potential to reduce the fungal overwintering inoculum by 80% to 90%.

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) μm long × (13-21) μm wide (n=30) in V8 juice agar, (22-38) μm long × (11-19) μm wide (n=30) in PCA, (21-41) μm long × (13-20) μm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 μm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

First report of Stemphylium leaf spot caused by Stemphylium vesicarium on alfalfa (Medicago sativa) in Peru

Alfalfa (Medicago sativa) is the most cultivated fodder crop in Peru with 172,000 ha cultivated (MINAM 2019), and Arequipa is the top producing region with 40% of the national production in 2015 (Santamaría et al. 2016). In January–April 2019 (av. 20°C and 70% RH), most alfalfa fields in Majes-Pedregal, Arequipa were affected by an unidentified foliar disease. One of the fields was located at the farm of the Universidad Nacional de San Agustín de Arequipa (16°19’29.6” S, 72°12’59.9” W). Symptoms appeared as elliptical light brown spots witdark brown borders (Fig. S1a and b). The field (~60 × 60 m) was divided into ~30 × 12 m sections and two plants in each section were collected (20 plants total). Plants were digitized and the leaflet diseased area was calculated with ImageJ 1.53a, from which an incidence of 100% and a severity of 38.7 ± 4.4 % were estimated. Microscopical observations at the leaflet spots revealed consistently the presence of oblong multiseptated conidia (23.6–42.8 × 16.5–25.2 µm; av. 33.3 × 20.9 µm; n = 40) of the genus Stemphylium (Simmons 1969; Woudenberg et al. 2017) (Fig. S1c). We obtained 10 pure cultures by placing conidia from the spots directly onto potato dextrose agar medium with the aid of stereoscope and sterile forceps. Two isolates (UNSA-StemV01 and UNSA-StemV02) were incubated further until ascospore production at room temperature with no special light stimulus. After 45 days of growth, globose pseudothecia and ellipsoidal ascospores (25.4–38.7 × 11.2–16.6 µm; av. 31.9 × 13.7 µm; n = 30) formation occurred (Fig. S1d and e). We extracted the DNA from these two isolates using Wizard® Purification Kit (Promega Corp., Madison, WI) and sequenced the internal transcribed spacer 1 and 2 intervening 5.8S rDNA subunit (GenBank accessions: MT371236–37), and the glyceraldehyde-3-phosphate dehydrogenase (MT375513–14) and the calmodulin (MT375515–16) genes, highly resolutive markers to identify Stemphylium species, following Woudenberg et al. (2017). We retrieved sequence data available from 43 isolates of nine Stemphylium species (Han et al. 2019; Woudenberg et al. 2017), and built a mid-point rooted phylogeny with the three-loci concatenated data set (Fig. S2). We identified our isolates as S. vesicarium (Fig. S2). Koch’s postulates were fulfilled by spray-inoculation with conidia from isolate UNSA-StemV01 suspended in sterile water (1×104 / mL) to two healthy 50-day old alfalfa plants growing on pots in the university greenhouse (av. 25°C and 70% RH). Two plants sprayed with sterile water without conidia served as control. Symptoms appeared after 21 days of inoculation, and when conidia were re-isolated, they were the same as originally obtained. No symptoms developed in the control plants. This confirmed that S. vesicarium is the causal agent of the alfalfa disease in Majes-Pedregal, identified as Stemphylium leaf spot. revious studies documented S. vesicarium on asparagus and onion in Peru (Castillo Valiente 2018; Vásquez Salas 2018; Vásquez Sangay 2013), but molecular characterization has only been applied to S. lycopersici from potatoes (Woudenberg et al. 2017). Stemphylium vesicarium has been documented in various crops, including alfalfa, and countries in Europe, North America, Africa, Asia and in Australia and New Zealand (Han et al. 2019; Woudenberg et al. 2017). This occurrence is the first report of S. vesicarium on alfalfa in Peru. The disease compromises the quality of this fodder crop, so actions need to be taken in Arequipa.

Stemphylium vesicarium. [Distribution map].

A new distribution map is provided for Stemphylium vesicarium (Wallr.) E.G. Simmons. Dothideomycetes: Pleosporales: Pleosporaceae. Hosts: many, including Allium spp., asparagus (Asparagus officinalis), pear (Pyrus sp.), barley (Hordeum vulgare), sunflower (Helianthus annuus). Information is given on the geographical distribution in Africa (Ethiopia, Egypt, Kenya, Libya, Senegal, South Africa, Tunisia, Zambia), Asia (Bhutan, Bangladesh, China, Hainan, Yunnan, India, Bihar, Haryana, Himachal Pradesh, Jammu and Kashmir, Karnataka, Maharashtra, Punjab, Rajasthan, Indonesia, Iran, Iraq, Israel, Japan, Hokkaido, Malaysia, Myanmar, Nepal, Pakistan, Saudi Arabia, Korea Republic, Sri Lanka, Taiwan, Turkey, United Arab Emirates), Europe (Belgium, Bulgaria, Cyprus, Denmark, France, Germany, Greece, Italy, Netherlands, Poland, Portugal, Russia, Slovakia, Spain, Sweden, Switzerland, Ukraine, United Kingdom, England), North America (Canada, Alberta, British Columbia, Manitoba, Nova Scotia, Ontario, Saskatchewan, Cuba, Jamaica, Mexico, USA, Arizona, California, Florida, Michigan, New Mexico, New York, Texas, Washington, Wisconsin), Oceania (Australia, New South Wales, Queensland, South Australia, Victoria, Western Australia, New Zealand, Tonga, Vanuatu), South America (Argentina, Bolivia, Brazil, Goiás, Chile, Colombia, Venezuela).