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.

Efficient fluorescence-based localization technique for real-time tracking endophytes route in host-plants colonization.

Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence-based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host-plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC-10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid-mediated transformation efficiencies of antibiotic-resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA-tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence-based technique's high resolution and reproducibility make it a platform-independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host-plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately 3 months.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. RESULTS: Regenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety 'Performer' tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines. CONCLUSION: The new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6-7 months from culture establishment.