Comparing the Fungicide Sensitivity of Sclerotinia sclerotiorum Using Mycelial Growth and Ascospore Germination Assays

The infection of the floral tissues of snap bean and other crops by Sclerotinia sclerotiorum, the causative agent of white mold, is by ascospores. Irrespective of the fungicide mode of action being evaluated, in vitro fungicide sensitivity tests are conducted almost exclusively using mycelial growth assays. This is likely due to difficulties and time involved in sclerotial conditioning required to produce apothecia and ascospores. The objective of this research was to compare estimates of fungicide sensitivity between mycelial growth and ascospore germination assays for S. sclerotiorum. Sensitivity assays were conducted using serial doses of three fungicides commonly used to control white mold: boscalid, fluazinam, and thiophanate-methyl. A total of 27 isolates were evaluated in replicated trials conducted for each fungicide and assay type. The effective concentration to reduce mycelial growth or ascospore germination by 50% (EC50) was estimated for each isolate, fungicide, assay type, and trial. The median EC50 values obtained from ascospore germination assays were 52.7, 10.0, and 2.7 times higher than those estimated from the mycelial growth for boscalid, fluazinam, and thiophanate-methyl, respectively. No significant correlation was found between EC50 values estimated by the two methods. These findings highlight differences that may be important in evaluating the sensitivity of S. sclerotiorum given the fungicide mode of action and how they will be used in the field.

Global Sexual Fertility in the Opportunistic Pathogen Aspergillus fumigatus and Identification of New Supermater Strains

A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits.

Antagonism of saprobe fungi from semiarid areas of the Northeast of Brazil against Sclerotinia sclerotiorum and biocontrol of soybean white mold

The antagonistic activity of 25 saprobe fungi from semiarid areas of Northeast Brazil was evaluated against Sclerotinia sclerotiorum (Lib.) de Bary (Helotiales: Sclerotiniaceae). Four fungi [Myrothecium sp. Tode (Hypocreales: Stachybotryaceae) isolate 2, Volutella minima Höhn. (Hypocreales: Nectriaceae), Phialomyces macrosporus P.C. Misra & P.H.B. Talbot (Pezizomycotina) and Dictyosporium tetraseriale Goh, Yanna & K.D. Hyde (Pleosporales: Dictyosporiaceae)] were selected and further tested their ability to inhibit mycelial growth, sclerotia formation and ascospore germination of S. sclerotiorum and to control white mold on soybean plants. V. minima and P. macrosporus filtrates at 50% effectively suppressed mycelial growth and Myrothecium sp. isolate 2 completely suppressed sclerotia formation and inhibited ascospore germination by over 95%, the same result as commercial fungicide fluazinam. Soybean plants pre-treated with Myrothecium sp. isolate 2, P. macrosporus, and V. minima and inoculated with S. sclerotiorum showed a reduction of 55.8%, 79.7%, and 83.2% of area under disease progress curve (AUDPC) of white mold, respectively, in relation to water. Collectively, these results underline the antagonistic activity of V. minima, P. macrosporus, and Myrothecium sp. isolate 2 against S. sclerotiorum and their potential as biocontrol agents of soybean white mold.

Effect of Wetness Duration and Incubation Temperature on Development of Ascosporic Infections by Sclerotinia sclerotiorum

The impact of wetness duration and incubation temperatures on Sclerotinia sclerotiorum ascospore germination and ascosporic infection efficiency were evaluated. Ascospore germination was optimal when incubated in continuous moisture (free water) at 21°C. Significantly lower germination was observed at 10 or 30°C. Interrupting ascospore wet incubation was detrimental for germination. In infection efficiency studies, dry bean and canola flowers were inoculated with dry ascospores and placed on leaves of dry bean and canola plants, respectively. Dry bean plants were incubated for 196 h at 18 to 20°C in alternating 8 to 16 h wet/12 to 24 h dry periods. Canola plants were incubated for 240 h at 10, 15, 20, 25, or 30°C in alternating 6 to 18 h wet/18 to 6 h dry periods. Interrupting wet incubation delayed symptom appearance and hindered development of the epidemics on both plant types. Logistic regression models estimated at 50% the probability of disease development on dry bean and canola plants when 68 and 48 h of wet incubation at 20°C accumulated in a period of 6 days, respectively. The canola model was validated using data from field trials. Results of these studies will contribute to develop more accurate warning models for diseases caused by S. sclerotiorum.

Inoculum and Infection Dynamics of Polystigma amygdalinum in Almond Orchards in Spain

Red leaf blotch (RLB) disease of almond, caused by Polystigma amygdalinum, is an important foliar disease in most production regions of the Mediterranean basin and the Middle East because severe infections may cause a premature defoliation of the tree. Some key aspects on the epidemiology of P. amygdalinum were studied in multiyear trials in two almond-growing regions in Spain, which included the seasonal development of perithecia and production and germination of ascospores along with the disease incubation and plant infectivity periods. Our results showed that primary inoculum was available in extended periods (January to August). Significant differences in ascospore amounts among regions, higher in the southern Andalusia and lower in the northern Catalonia, and years of study were detected. The factors geographical location, sampling period, and evaluation year were found significant on the development of P. amygdalinum perithecia. Variable ascospore germination rates were observed from April to July: >15% but rarely exceeding 30%. The RLB infectivity period in Catalonia extended from March to mid-June, whereas in Andalusia it was from March to May. The incubation period was mainly in a range of 5 to 10 weeks in Catalonia. The environmental conditions of October to January influence the available ascospore amounts in the next season. RLB infection occurs in spring to summer when mean temperatures are in the range 10 to 20°C. These results represent the first step in developing a prediction model of the disease that might serve as a tool for the control of RLB.

Phyllachora maydis Ascospore Release and Germination from Overwintered Corn Residue

Tar spot of corn, caused by Phyllachora maydis, has been reported in several upper Midwest states in the United States. This has led to expanded efforts to more thoroughly understand the biology of P. maydis and the epidemiology of tar spot. This study determined the potential for P. maydis ascospore release and germination from overwintered P. maydis-infected corn residues from various locations in the upper Midwest. Corn residue samples collected in the spring of 2019 from 12 fields in four states were examined. Ascospore release and germination were observed in all residue samples collected. The mean total number of ascospores released per milliliter of water ranged from 3.6 × 103 to 4.8 × 106 after 4-h incubation and 3.7 × 103 to 4.4 × 106 after 24-h incubation. The mean percent spore germination ranged from 0.7 to 24.1% after 4-h incubation and 2.4 to 24.9% after 24-h incubation. There was a significant inverse relationship between total numbers of ascospores released and the percent germination of the ascospores. Samples from Illinois consistently yielded the greatest total ascospore release but were also consistently among the samples with the lowest percent ascospore germination. Samples from Wisconsin and Indiana were among the lowest for total ascospore numbers but were among the highest for total ascospore germination. These findings provide evidence that P. maydis can overwinter in multiple areas of the upper Midwest. Future research should focus on reducing infested corn residue to reduce initial infection by P. maydis.

A case study on the re-establishment of the cyanolichen symbiosis: where do the compatible photobionts come from?

Background and AimsIn order to re-establish lichen symbiosis, fungal spores must first germinate and then associate with a compatible photobiont. To detect possible establishment limitations in a sexually reproducing cyanolichen species, we studied ascospore germination, photobiont growth and photobiont association patterns in Pectenia plumbea.MethodsGermination tests were made with ascospores from 500 apothecia under different treatments, and photobiont growth was analysed in 192 isolates obtained from 24 thalli. We determined the genotype identity [tRNALeu (UAA) intron] of the Nostoc cyanobionts from 30 P. plumbea thalli from one population. We also sequenced cyanobionts of 41 specimens of other cyanolichen species and 58 Nostoc free-living colonies cultured from the bark substrate.Key ResultsNot a single fungal ascospore germinated and none of the photobiont isolates produced motile hormogonia. Genetic analyses revealed that P. plumbea shares Nostoc genotypes with two other cyanolichen species of the same habitat, but these photobionts were hardly present in the bark substrate.ConclusionsDue to the inability of both symbionts to thrive independently, the establishment of P. plumbea seems to depend on Dendriscocaulon umhausense, the only cyanolichen species in the same habitat that reproduces asexually and acts as a source of appropriate cyanobionts. This provides support to the hypothesis about facilitation among lichens.

Germination of Fusarium graminearum Ascospores and Wheat Infection are Affected by Dry Periods and by Temperature and Humidity During Dry Periods

The effects of temperature and relative humidity (RH) on germination of Fusarium graminearum ascospores, and of dry periods (DP) of different lengths and of temperature and RH during DP on ascospore survival were studied both in vitro and in planta. Optimal temperatures for ascospore germination at 100% RH were 20 and 25°C; germination was ≤5% when ascospores were incubated at 20°C and RH ≤ 93.5%. Viable ascospores were found at all tested combinations of DP duration (0 to 48 h) × temperature (5 to 40°C) or RH (32.5 to 100% RH). Germination declined as DP duration and temperature increased. Germination was lower for ascospores kept at 65.5% RH during the DP than at 76.0, 32.5, or 93.5% RH. Equations were developed describing the relationships between ascospore germination, DP duration and temperature or RH during DP. Durum wheat spikes were inoculated with ascospores and kept dry for 0 to 48 h at approximately 15°C and 65% RH; plants were then kept in saturated atmosphere for 48 h to favor infection. Fungal biomass, measured as F. graminearum DNA by quantitative polymerase chain reaction, declined as DP increased to 24 and 48 h at 3 and 9 days postinfection but not in spikes at maturity.