First report of root rot of strawberry caused by Fusarium cugenangense, a member of the F. oxysporum species complex, in Tennessee, USA.

Strawberry (Fragaria × ananassa Duch) in Tennessee is cultivated on plastic mulched beds annually, and production is limited primarily by multiple oomycete and fungal root rot pathogens that result in reduced vigor and black root rot disease symptoms. In early June 2018, plants (cv. Chandler) with reduced shoot vigor and size, and black, necrotic stunted roots were collected from Rhea County, TN. Roots and crowns of 10 plants were cut into 1-3 cm pieces and surface sterilized with 0.6% NaOCl, followed by 70% ethanol for 1 min each, and plated on water agar. White mycelia produced after 3 days were transferred to potato dextrose agar amended with 10 mg/liter rifampicin. After 10 days, fungal colonies were light purple on the surface and dark purple on the colony underside, later developing blue-black pigmentation on the underside. Microconidia on carnation leaf agar were ovoid to ellipsoid, aseptate or septate and 8.0 to 24.2 (13.7) × 3.0 to 4.5 (3.8) µm in size, macroconidia were 3 to 5 septate and falcate to almost straight and 33.7 to 52.8 (44.4) × 4.0 to 5.5 (4.9) µm in size (n=80); both conidia were produced on monophialides. Chlamydospores were globose and subglobose, formed terminally and intercalary on aerial, submerged, and surface mycelium, singly or in pairs and were abundantly produced in sucrose broth and on synthetic nutrient-poor agar (SNA) (diam. 7.6 µm). Morphology was consistent with Fusarium oxysporum (Leslie and Summerell, 2006) and F. cugenangense, a member of the F. oxysporum species complex, as described by Maryani et al. (2019). Fungal mycelia were used for PCR (Phire Plant Direct PCR Master Mix, Thermo Scientific, CA) and the translational elongation factor 1-α (EF1α) region was amplified with primers EF-1/EF-2 (O'Donnell et al., 1998), internal transcribed spacer (ITS) regions amplified with primers ITS1/ITS2 (White et al. 1990), and the RNA polymerase second largest subunit region (RPB2) with primer pairs 5f2/7cr and 7cf/11ar (O'Donnell et al., 2022). PCR products of isolate SC5 were sequenced, and sequences compared to all sequences in the FUSARIOID-ID database using polyphasic identification (Crous et al., 2021) with EF1α (GenBank Accession No. ON703236) and RPB2 (OR472390) sequences. The highest similarity (100%) was with isolates of F. cugenangense, including ex-type isolate InaCC F984 (99.94% similarity) (Maryani et al., 2019). F. cugenangense is closely related to F. callistephi and F. elaeidis, but both species lack chlamydospores, and F. elaeidis has polyphialides (Lombard et al, 2019). To satisfy Koch's postulates, healthy rooted strawberry plants produced in soilless media were transplanted into 4 plastic pots (1.2-liter) containing 5% (w/v) fungal inoculum (grown on barley grain) and mixed into the top 5-cm of peat-based soilless medium. Pots were incubated at 25°C and 50% RH in a growth chamber. Four pots without inoculum served as controls. The trial was repeated. Within 8 weeks, all inoculated plants had low vigor, with necrotic and stunted roots. Root sections of control and inoculated plants were plated, and the pathogen was re-isolated from diseased roots of all inoculated plants only and confirmed as F. cugenangense based on morphology and sequence analysis. To our knowledge, this is the first report of F. cugenangense, or any member of the F. oxysporum species complex, causing root rot of strawberry in Tennessee and could be an important component of the production-limiting black root rot disease complex of strawberry.

First Report of Strawberry Black Root Rot Caused by Globisporangium sylvaticum in Tennessee, USA.

Globisporangium sylvaticum (syn. Pythium sylvaticum), is an oomycete that causes root rot and damping off of field crops, ornamentals, and vegetables. Several species in Pythiaceae are associated with black root rot of strawberry [(Fragaria × ananassa) Duchesne] (Millner 2006). Mature, stunted 'Chandler' strawberry plants, with reduced shoot vigor and black necrotic roots, were collected from Rhea County (June 2018) and Cumberland County, TN (May 2019). Aboveground symptoms occurred in low incidence (<5% of plants) in the fields. Plant roots were rinsed with tap water, cut into 1 to 3 cm pieces, and surface-disinfested (70% ethanol, 1 min) followed by a sterile water rinse. Root segments were crushed, placed on 20% V8 juice agar, and incubated in the dark at 21°C for 3 days. White fluffy mycelia grew from a majority of roots and coenocytic hyphae with globose hyphal swellings, delimited from hyphae by septa, were observed with microscopy. Hyphae were initially branched, curled, hyaline, and aseptate; however, septations were observed in older cultures. Globose structures (terminal and intercalary) were identified as sporangia [11 to 32 (avg. 22.1) µm diameter] when zoospores were observed (Parikh et al. 2022). Oospores [9 to 21 (avg. 16) µm diameter] were globose, smooth, aplerotic, and thick-walled. Oogonia, with or without one or more inflated antheridia, were observed when isolates were paired in culture, characteristics consistent with descriptions of Campbell and Hendrix (1967), Pratt and Green (1971), van der Plaats-Niterink (1981), and Uzuhashi et al. (2010). Genomic DNA was extracted (Extract-N-Amp™; Sigma-Aldrich, MO) for PCR amplification of internal transcribed spacer (ITS) regions of rDNA with primers ITS1/ITS4 (White et al. 1990); ITS and large subunit rRNA regions with primers UN-up18S42/UN-lo28S22 (Robideau et al. 2011); and cytochrome c oxidase subunit I (COI) mitochondrial DNA with primers OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011). Primers ITS1/ITS4 were used to amplify isolate TN (GenBank Accession MW386310, which had 100% homology with reference isolate MK326528). Primers UN-up18S42/UN-lo28S22 amplified isolates SAP18 and OO1 (Accessions MZ881935 and MZ881936, which had 99.8% homology with HQ665236), and COI primers amplified isolate SAP18 (Accession OK020192, which had 100% homology with GU071816 and KT692835). To satisfy Koch's postulates, inoculum of G. sylvaticum grown on autoclaved wheat seeds was added (5% w/v) to planting mix (1 peat:1 sand, v/v). Young, rooted strawberry plants were planted in 1.2-L pots with infested (n = 6) and control (no pathogen, n = 6) mixes, which was saturated with deionized water. Pots were covered with clear plastic for 48 h to maintain high humidity. Plants were grown in a greenhouse (24°C avg.) for 8 weeks. The disease assay was repeated. All plants in infested mix died, with black, necrotic roots. Plants in the control mix were healthy and well-established. The pathogen was reisolated from roots of all inoculated plants and confirmed to be G. sylvaticum based on morphology and molecular analyses. Root disease of strawberry caused by G. sylvaticum has been reported in the USA (Campbell and Hendrix 1967; Nemec and Sanders 1970; Pratt and Green 1971). This is the first report of G. sylvaticum causing root rot of strawberry in Tennessee. With the loss of methyl bromide, sustainable disease control strategies are needed to provide effective management options for strawberry black root rot.

Anaerobic Soil Disinfestation Efficacy Against Fusarium oxysporum Is Affected by Soil Temperature, Amendment Type, Rate, and C:N Ratio.

A meta-analysis of anaerobic soil disinfestation (ASD) efficacy against Fusarium oxysporum and F. oxysporum f. sp. lycopersici was conducted emphasizing effects of environment and organic amendment characteristics and pot and field studies conducted on ASD amendment C:N ratio and soil temperature effects on F. oxysporum f. sp. lycopersici inoculum survival. In a pot study, two organic amendments, dry molasses-based or wheat bran-based, applied at 4 mg of C/g of soil, with 40:1, 30:1, 20:1, and 10:1 C:N ratios, were evaluated against F. oxysporum f. sp. lycopersici at 15 to 25°C. This study was followed by a pot study with temperature regimes of 15 to 25°C and 25 to 35°C and two C:N ratios (20:1 and 40:1), and a field study at 40:1, 30:1, 20:1, and 10:1 C:N ratios, a 30:1 C:N ratio at a lower C rate (2 mg of C/g of soil), and an anaerobic control. Soil temperature >25°C and more labile amendments increased ASD suppression of F. oxysporum/F. oxysporum f. sp. lycopersici in the meta-analysis. In pot studies, F. oxysporum f. sp. lycopersici survival was reduced for molasses-based mixtures at 20:1 and 30:1 C:N ratios compared with wheat bran-based mixtures but not compared with the anaerobic control. At 25 to 35°C, all ASD treatments suppressed F. oxysporum f. sp. lycopersici relative to controls. In the field, all ASD treatments reduced F. oxysporum f. sp. lycopersici survival compared with the anaerobic control, and 4 mg of C/g of soil amendment rates induced higher anaerobic conditions and higher F. oxysporum f. sp. lycopersici mortality compared with the 2 mg of C/g of soil rate. Although amendment C:N ratios from 10 to 40:1 were similarly suppressive of F. oxysporum, lower temperatures reduced ASD effectiveness against F. oxysporum/F. oxysporum f. sp. lycopersici and further work is warranted to enhance suppression at soil temperatures <25°C.

Anaerobic Soil Disinfestation Reduces Germination and Affects Colonization of Sclerotium rolfsii Sclerotia.

Growth chamber and field studies were conducted with organic amendment mixtures of carbon (C) and nitrogen (N) at C:N ratios 10:1, 20:1, 30:1, and 40:1 and amendment rates of C at 2, 4, 6, and 8 mg/g of soil (C:N ratio 30:1) to evaluate anaerobic soil disinfestation (ASD) effects on germination and colonization of Sclerotium rolfsii. In the growth chamber, sclerotial germination was reduced in all ASD treatments regardless of C:N ratio (0.6 to 8.5% germination) or amendment rate (7.5 to 46%) as compared with nonamended controls (21 to 36% and 61 to 96%, respectively). ASD treatment increased Trichoderma spp. colonization of sclerotia, with consistently higher colonization in ASD treatments with amendment rates of C at 2 or 4 mg/g of soil (>87% colonization) compared with nonamended controls (<50% colonization). In the 2014 field study, sclerotial germination was reduced by 24 to 30% in ASD treatments when compared with the nonamended control. Sclerotial colonization by Trichoderma spp. was predominant; however, other potential mycoparasites (i.e., Aspergillus spp., Fusarium spp., zygomycetes, and other fungi) were present in the field study. Amendment C:N ratios in the range of 10:1 to 40:1 were equally effective in reducing sclerotial germination and enhancing colonization by potentially beneficial mycoparasites of sclerotia.

A Meta-Analysis of the Impact of Anaerobic Soil Disinfestation on Pest Suppression and Yield of Horticultural Crops.

Anaerobic soil disinfestation (ASD) is a proven but relatively new strategy to control soil borne pests of horticultural crops through anaerobic decomposition of organic soil amendments. The ASD technique has primarily been used to control soil borne pathogens; however, this technique has also shown potential to control plant parasitic nematodes and weeds. ASD can utilize a broad range of carbon (C) amendments and optimization may improve efficacy across environments. In this context, a meta-analysis using a random-effects model was conducted to determine effect sizes of the ASD effect on soil borne pathogens (533 studies), plant parasitic nematodes (91 studies), and weeds (88 studies) compared with unamended controls. Yield response to ASD was evaluated (123 studies) compared to unamended and fumigated controls. We also examined moderator variables for environmental conditions and amendments to explore the impact of these moderators on ASD effectiveness on pests and yield. Across all pathogen types with the exception of Sclerotinia spp., ASD studies show suppression of bacterial, oomycete and fungal pathogens (59 to 94%). Pathogen suppression was effective under all environmental conditions (50 to 94%) and amendment types (53 to 97%), except when amendments were applied at rates less than 0.3 kg m(-2). The ASD effect ranged from 15 to 56% for nematode suppression and 32 to 81% for weed suppression, but these differences were not significant. Significant nematode moderators included study type, soil type, sampling depth, incubation period, and use of mixed amendments. Weed suppression due to ASD showed significant heterogeneity for all environmental conditions, confirming that these studies do not share a common effect size. Total crop yield was not reduced by ASD when compared to a fumigant control and yield was significantly higher (30%) compared to an unamended control, suggesting ASD as a feasible option to maintain yield without chemical soil fumigants. We conclude ASD is effective against soil borne pathogens and while not conclusive due to a limited number of studies, we expect the same for nematodes and weeds given observed effect sizes. Findings should assist researchers in exploring ASD efficacy in particular environmental conditions and allow for development of standard treatment protocols.