The microbial ecology of agricultural products may provide crucial insights into the management of post-harvest fruit rots. To investigate post-harvest microbial communities of highbush blueberry (Vaccinium corymbosum), five fungicide spray programs were evaluated for their influence on the mycobiome of fruit skin and within the fruit pulp. The mycobiome was characterized by sequencing amplicons of the ITS1 region with primers ITS1f and ITS4 with the Illumina MiSeq 300bp v3 system. Two of the five programs utilized commercial biological fungicides, two utilized azoxystrobin, and one utilized a series of treatments to simulate a realistic disease management program. Fungicide applications reduced diversity of the fruit skin mycobiome (R2=0.409, p=0.0001) and had a moderate impact on the pulp mycobiome (R2=0.233, p=0.0001). The mycobiome of the fruit pulp was also more variable than the skin mycobiome. In comparison to the untreated controls, each fungicide treatment program had a strongly significant effect on the beta-diversity of the blueberry fruit skin mycobiome (R2=0.53-0.73, p=0.0001). In the pulp, three of the five treatments had moderate but significant effects on beta-diversity in comparison to the control (R2=0.10-0.18, p=0.0005-0.017). Most samples indicated that fungi belonging to Epicoccum, Papiliotrema, and Sporobolomyces were widely prevalent and abundant across treatments and tissues. Fruit pathogen Botrytis cinerea was particularly abundant in the pulp of three of fungicide treatments. Results from this study provide a baseline for future exploration of post-harvest rot pathology and provide a community context on how fungicides may alter fungal communities in agricultural systems.
Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs, including pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen with commonly used SDHIs, boscalid and fluopyram. Different SdhB mutations were detected in a B. cinerea collection from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I at frequencies 82.6, 4.3, 11.5, 0.4, and 5.3 %, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We have classified the resistant profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤ 10 pg, and in a single assay was capable of monitoring two amino acids positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.
In July 2020, a 3-year-old ‘Envoy’ northern highbush blueberry bush (Vaccinium corymbosum L.) from a commercial farm in Van Buren County, Michigan was submitted to the Plant & Pest Diagnostics laboratory at Michigan State University. Field disease assessments across the 2-acre planting were an incidence of 2-5% and a severity of 50-100%. Symptoms included red shoot flagging and dead shoots retaining dry leaves, shoots with light green leaves and necrotic margins, and brown-black cankers at the base of the symptomatic shoots. Shoot sections displaying wood discoloration were surface disinfested by dipping in 95% ethanol and flame sterilized. The internal discolored tissues (0.5 cm2) were plated onto 1% ampicillin and streptomycin, quarter-strength potato dextrose agar (PDA) and incubated at room temperature until fungal colonies were observed. A fungus with rapid growth, developing white and then dark gray mycelium, resembling species in the family Botryosphaeriaceae was isolated and subcultured. After DNA extraction and amplification, sequences of three loci were obtained: the internal transcribed spacer (ITS) region, β-tubulin (Bt), and elongation factor 1-α (EF1) using primer pairs ITS1/ITS4, Bt2a/Bt2b, and EF1-728/EF1-986R, respectively (Slippers et al. 2004). The sequences showed 100% identity with Genbank numbers KF766205 (ITS region, 562 bp – OP588109), MT592721 (Bt, 436 bp – OP585548), and MT592229 (EF1, 306 bp – OP585547) of N. ribis (Slippers et al. 2013, Zhang et al. 2021). Sequences were identified using PopSet 1995604550 (Zhang et al. 2021). Pathogenicity was tested on 2-year-old ‘Blueray’ blueberries. Five plants, 3 shoots per plant (n = 15) were surface-sterilized with a 3% bleach solution by rinsing, wounded with razor blades using a scratching method in ‘X’ patterns across the length of the wound, and then inoculated using mycelium plugs (5 mm) from 7-day old cultures grown on full-strength PDA. Plugs were crushed and spread onto the wound and the wound was wrapped with parafilm. Control plant shoots (n = 9) were mock inoculated using sterile PDA plugs. Plants were maintained in the greenhouse at 23°C under a 14-hour photoperiod and imaged at 0-, 7-, and 12-days post inoculation (dpi). Symptoms began developing within 7 dpi. At 12 dpi, 9 of 15 inoculated shoots began displaying leaf necrosis and deep red or brown stem discoloration 6 cm above and below the wound, while controls remained healthy. Fungi morphologically identical to the original isolate were reisolated from sections taken from 2.5, 6.3, and 7 cm above and below the inoculation site. Species identity was confirmed by sequencing as described above. Neofusicoccum species are widespread and commonly associated with canker and dieback symptoms of blueberries (Flor et al. 2022). To our knowledge, this is the first report of stem blight and dieback caused by N. ribis in Michigan blueberry production. The species N. parvum and N. ribis have been reported on southern highbush blueberries in California (Koike et al. 2014) and Florida (Wright and Harmon 2010), but neither has been reported on blueberry in Michigan. Accurate diagnoses of Botryosphaeria fungal species in blueberries is critical for effective disease control and yield loss reduction.
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