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.

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.

Figure 4 Figure 5 Cowpea (Vigna unguiculata) is a productive vegetable, grain, forage or cover crop legume that is particularly adapted to subtropical and tropical climates where stresses such as drought, high temperature and marginal soil conditions are common. In 2014, several genotypes of surface-sterilised cowpea seeds were grown in a greenhouse at the University of Tennessee, Knoxville and after eight weeks, plants showed signs of wilting and necrosis of stems and leaves in concurrence with dark fungal growth (Fig. 1). Concurrently, seeds from the same lots (grown in Jackson, TN) were surface sterilised for one minute with 100 ml/l bleach (active ingredient 6% NaOCl), rinsed with sterile water, and plated onto water agar (WA). Cowpea seed produced white-to-grey mycelia that darkened upon maturation and were visually similar to the fungus observed in the greenhouse trial (Fig. 2). The fungus was isolated on WA, transferred to potato dextrose agar, and incubated until sporulation (Fig. 3).

Cowpea (Vigna unguiculata) is a productive vegetable, grain, forage or cover crop legume that is particularly adapted to subtropical and tropical climates where stresses such as drought, high temperature and marginal soil conditions are common. In 2014, several genotypes of surface-sterilised cowpea seeds were grown in a greenhouse at the University of Tennessee, Knoxville and after eight weeks, plants showed signs of wilting and necrosis of stems and leaves in concurrence with dark fungal growth (Fig. 1). Concurrently, seeds from the same lots (grown in Jackson, TN) were surface sterilised for one minute with 100 ml/l bleach (active ingredient 6% NaOCl), rinsed with sterile water, and plated onto water agar (WA). Cowpea seed produced white-to-grey mycelia that darkened upon maturation and were visually similar to the fungus observed in the greenhouse trial (Fig. 2). The fungus was isolated on WA, transferred to potato dextrose agar, and incubated until sporulation (Fig. 3). Morphological characteristics were compared to descriptions of Diplodia seriata (Phillips et al., 3). Conidia were 26.5 × 11.9 μm (n = 100), hyaline, tinted to dark brown, and oval to ellipsoidal. Most conidia were aseptate, a few had one septum, which has been reported for this species (Phillips et al., 4). Conidia were borne singly on conidiophores inside dark brown, globose, ostiolate pycnidia. Perithecia had asci that contained eight hyaline, aseptate, fusoid ascospores (Fig. 4). Molecular identification was performed with genomic DNA (Extract-N-Amp“ Kit, Sigma-Aldrich, USA) of fungal mycelia, PCR amplification of internal transcribed spacer (ITS) regions with primers ITS1 and ITS4 (White et al., 5), followed by amplicon sequencing. Sequences obtained shared 100% identity with several accessions of D. seriata (e.g. GenBank Accession Nos. MH675476.1, KY385656.1 and HQ660463.1). The amplicon sequence was deposited in GenBank (MK257126). For initial pathogenicity assays, actively growing mycelial plugs from WA were transferred onto cowpea leaves on moist filter paper in lidded Petri dishes. Symptoms on leaves of cowpea cv. ‘Early Acre’ included yellowing, and pinpoint dark spots that expanded to lesions with a light brown centre and dark brown, irregular-shaped margins (Fig. 5). To fulfil Koch's postulates, the fungus was produced on oat inoculum and a conidial suspension was prepared from 300 g inoculum in 200 ml sterile water. Leaves and stems of three-week-old plants of cowpea cv. ‘Early Acre’ (n = 10) were sprayed with the conidial suspension. Inoculated and control plants (n = 10, sprayed with sterile water) were individually covered with clear plastic bags for four days, after which they were uncovered and incubated in a growth chamber for three weeks at 25°C. Leaf discolouration and wilting were evident in all inoculated plants in the first trial after one week, and in eight of ten plants in the second trial. Fungal samples from infected leaves were re-isolated and morphological and molecular identification were carried out as described above. The re-isolated fungus had 100% identity to D. seriata. Diplodia seriata infects a wide range of woody plant species with varying severity (Díaz et al., 1; Elena et al., 2; Phillips et al., 4) and in the case of cowpea, the pathogen can affect both leaf and stem tissue causing wilt, discolouration, and significant leaf loss. To our knowledge, this is the first report of infection of cowpea by D. seriata.

HomePlant DiseaseVol. 104, No. 5First Report of Chaetomium globosum Causing a Leaf Spot of Hemp (Cannabis sativa) in Tennessee PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Chaetomium globosum Causing a Leaf Spot of Hemp (Cannabis sativa) in TennesseeA. G. Chaffin, M. E. Dee, S. L. Boggess, R. N. Trigiano, E. C. Bernard, and K. D. GwinnA. G. ChaffinDepartment of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, M. E. DeeDepartment of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, S. L. BoggessDepartment of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, R. N. Trigianohttp://orcid.org/0000-0002-7264-1822Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, E. C. BernardDepartment of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, and K. D. Gwinn†Corresponding author: K. D. Gwinn; E-mail Address: [email protected]http://orcid.org/0000-0002-3517-3433Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996AffiliationsAuthors and Affiliations A. G. Chaffin M. E. Dee S. L. Boggess R. N. Trigiano E. C. Bernard K. D. Gwinn † Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996 Published Online:17 Mar 2020https://doi.org/10.1094/PDIS-08-19-1697-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Cultivation of hemp (Cannabis sativa), a crop grown for food, fiber, biofuel, and natural medicine worldwide, has increased in the southeastern United States. Pilot and research programs were legalized in 2014, and hemp was removed from the list of Schedule I controlled substances in 2018 (Agriculture Improvement Act of 2018; Mead 2019). Little is known about hemp pathogens, but pathogens of marijuana strains grown in controlled environments were recently described (Punja et al. 2019). Symptomatic plants were found in the North Greenhouse at the University of Tennessee, Knoxville, TN, in September 2017. Leaf spots began as chlorotic lesions near the margins and progressed to necrotic lesions with chlorotic halos. Leaves with chlorotic and necrotic lesions were collected from three 4-month-old plants of two fiber hemp cultivars, Fedora 17 and Futura 75. Leaves were surface sterilized in a 6.7% sodium hypochlorite solution for 1 min, rinsed in deionized water, and incubated in sterile moist chambers for 1 week at 25°C. Mycelia that emerged from or near the lesions were transferred to potato dextrose agar (PDA), and incubated at 28°C. Mycelium was initially white but darkened to yellowish-green (B40C10Y80 [Küppers 1982]) after 1 month. Genomic DNA was amplified using Phire Plant Direct PCR Master Mix (ThermoScientific, Waltham, MA) according to the manufacturer's protocol utilizing ITS1 and ITS4 (White et al. 1990) and sequenced. For three isolates (HL1, HL3, and HL4), the sequence was a 100% match with Chaetomium globosum (GenBank accession no. KX421415.1) and deposited in GenBank (accession nos. MN233699, MN233700, and MN175258, respectively). Morphological characters were consistent with C. globosum (Wang et al. 2016). Ascomata (n = 10) had a mean length of 280 μm (240 to 313 μm) and width of 208 μm (174 to 233 μm). Ascospores were limoniform and olivaceous gray-brown (Y90M70C80 [Küppers 1982]) and had a mean length of 9.3 μm (8.7 to 10.9 μm) and width of 7.5 μm (6.8 to 8.1 μm) (n = 20). Isolate HL4 was cultured on PDA, and after 1 month, mycelial plugs (6 mm in diameter) or sterile PDA plugs were placed on symptom-free leaves of Fedora 17, Futura 75, and Wife, a high-cannabidiol cultivar. For detached leaf assays, eight leaves (three plugs per leaf) of each cultivar were inoculated; for whole plant assays, leaves of three plants of each cultivar (three plugs per leaf) were inoculated and were covered with plastic bags to increase humidity for fungal growth and sporulation. Symptoms identical to the original symptoms developed on Futura 75 and Fedora 17 leaves after a week, and reisolation on water agar amended with rifampicin (0.1 g/liter) confirmed the causal agent as C. globosum. There were no disease symptoms on inoculated Wife leaves or controls. C. globosum is an endophyte (Punja et al. 2019), a saprophyte, and a pathogen of plants (Guo et al. 2016) and humans (Wang et al. 2016); it also causes allergenic responses. It is a well-known biocontrol agent (reviewed in Aggarwal 2015). Chaetomium succineum was described as the causal agent of a leaf spot on C. sativa, but because C. succineum is now placed in the genus Arxotrichum (Crous et al. 2018), this is the first report of an undoubted species of Chaetomium causing disease on hemp. The disease may reduce the quantity of hemp products and cause economic loss to growers. Understanding diseases of hemp is an essential first step for developing successful management practices.The author(s) declare no conflict of interest.References:Aggarwal, R. 2015. Indian Phytopathol. 68:8. Google ScholarAgriculture Improvement Act of 2018. 2018. Page 420: in https://www.congress.gov/bill/115th-congress/house-bill/2/text. Google ScholarCrous, P. W., et al. 2018. Persoonia 40:240. https://doi.org/10.3767/persoonia.2018.40.10 ISI, Google ScholarGuo, J.-W., et al. 2016. Plant Dis. 100:223. https://doi.org/10.1094/PDIS-03-15-0243-PDN Link, ISI, Google ScholarKüppers, H. 1982. The Basic Law of Color Theory. Barrons Educational Series, Woodbury, NY. Google ScholarMead, A. 2019. Front. Plant Sci. 10:697. https://doi.org/10.3389/fpls.2019.00697 Crossref, Google ScholarPunja, Z., et al. 2019. Front. Plant Sci. 10:1120. https://doi.org/10.3389/fpls.2019.01120 Crossref, ISI, Google ScholarWang, X. W., et al. 2016. Persoonia 36:83. https://doi.org/10.3767/003158516X689657 Crossref, ISI, Google ScholarWhite, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Crossref, Google ScholarThe author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 104, No. 5 May 2020SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionSymptoms observed in the field on zucchini plants caused by Fusarium solani f. sp. cucurbitae (A. Pérez-Hernández et al.). Photo credit: J. M. Gómez-Vázquez. Peach tree with excavated root collar (S. B. Miller et al.). Photo credit: G. Schnabel. Metrics Article History Issue Date: 3 May 2020Published: 17 Mar 2020Accepted: 12 Jan 2020 Page: 1540 Information© 2020 The American Phytopathological SocietyKeywordshempCannabisChaetomiumbiodegradationleaf spotThe author(s) declare no conflict of interest.Cited byFirst Record of Chaetomium globosum Causing Leaf Spot of Pomegranate in PakistanM. W. Alam, A. Malik, A. Rehman, A. Hameed, T. A. Chohan, M. Sarwar, R. Mushtaq, M. Hussain, D. Hussain, and T. Shafeeq12 September 2021 | Plant Disease, Vol. 105, No. 8Development of a Co-Dominant Cleaved Amplified Polymorphic Sequences Assay for the Rapid Detection and Differentiation of Two Pathogenic Clarireedia spp. Associated with Dollar Spot in Turfgrass27 July 2021 | Agronomy, Vol. 11, No. 8

Anaerobic soil disinfestation (ASD; also termed biological soil disinfestation) is a non-chemical process which includes 1) soil incorporation of a labile carbon (C) source, 2) mulching with polyethylene film to limit gas exchange, and 3) drip irrigation to saturation of the topsoil or bedded area. A number of putative mechanisms have been proposed as contributing to control of pathogens, nematodes, and weeds during ASD treatment, although not all have been well-characterized. Mechanisms include formation of organic acids and volatile compounds during anaerobic decomposition of the added C source, biocontrol by microorganisms favored by ASD treatment, and changes in soil chemical constituents under anaerobic conditions. In Tennessee, USA, growth chamber, greenhouse, and field studies have been conducted to evaluate and optimize the ASD procedure for regional production systems and to evaluate pest, soil, and crop responses resulting from differing C source rates and properties. A growth chamber study conducted using soil temperatures typical to spring soil disinfestation treatment in this region (15 to 24°C), suggests that C amendment rates less than 1 mg C g-1 soil for ASD treatment do not consistently decrease viability of Sclerotinia sclerotiorum sclerotia or decrease incidence of endemic Fusarium root rot of common bean compared to an unamended control. Variability in measures of accumulated anaerobic soil conditions and an observed soil pH increase in ASD-treated soils are also indicative of ineffective ASD treatment at low amendment rates. Other preliminary work suggests that amendment rates may need to be as high as 4 mg C g-1 soil for effective soil disinfestation at moderate soil temperatures. Studies to determine optimal amendment rates and properties for consistent ASD treatment at moderate soil temperatures are ongoing.