Role of Local Trichoderma spp. Isolates in Reducing Tomato Fusarium Wilt and Increasing Phosphorus Uptake

Methodology Figures for SARE Grant_CaitlinDalton

1a: Using a randomized complete block design, we will evaluate biocontrol and plant growth promoting effects of eight indigenous Trichoderma isolates (Table 1) in a greenhouse trial in soil inoculated with F. oxysporum f. sp. lycopersici (Fol). Non-Trichoderma inoculated controls with and without Fol inoculation will be included. There will be four replicates of each treatment and the study will be repeated. Fol will be transferred from Potato Dextrose Agar (PDA) to be cultured on sterilized organic red wheat (ORW) to create inoculum for the study. Vials containing 150g of ORW will be inoculated with Fol using colonized plugs of PDA and stored under supplemental fluorescent lighting for 10-14 days until dense, white conidial growth is present (Shrestha, 2020). Trichoderma will be cultured on PDA for 5-7 days, and those with dense, green conidial growth will be used to create spore suspensions. Suspensions will be diluted to achieve spore concentrations of 1 x 10^6 spores/mL (Shrestha, 2020). Tomato seeds (cv. Valencia) will be surface sterilized with 3% hydrogen peroxide before soaking in each Trichoderma treatment conidial suspension, with seeds for the control soaked in sterile water. Seeds will then be planted in organic media and grown in a greenhouse for 3-4 weeks before transplanting to experimental pots. Experimental pots will consist of 10 different treatments with one of eight Trichoderma or a control (control 2) inoculated with Fol, in comparison to a control (control 1) with no Trichoderma or Fol treatment.  Fol inoculum will be applied at a rate of 2 g/pot prior to covering with a final layer of media. Plants will grow for 9 weeks.  Disease ratings will be recorded on a 1-4 scale (1= 1-25%, 2= 26-50%, 3= 51-75%, and 4= 75-100%) once a week from weeks 6 to 9 (post transplanting). After 9 weeks, height (cm) and stem caliper (mm) will be recorded. Tomatoes will be cleaned, and roots separated from shoot tissue. Tomato root systems will be assayed for colonization by Trichoderma by plating on Trichoderma selective media (TSM) (Askew and Laing, 1993; Williams et al., 2003).

1b: In lab trials, we will evaluate potential solubilization of Al- and Ca- phosphates by Trichoderma isolates, and evaluate how P solubilization potential by Trichoderma isolates impacts interactions with Fol in dual culture plate assays. Trichoderma isolates (Table 1) and Fol will be cultivated on PDA until sporulation occurs. Pikovskaya’s Media (PVK) will be prepared with three varying phosphorus sources at a single P rate: aluminum phosphate (AlPO₄), mono-potassium-phosphate as a highly soluble positive control, and tri-calcium phosphate [Ca₃(PO₄)₂]; and one no phosphorus (negative) control treatment (Rinu et al., 2013 & Doilom et al., 2020). Five-mm diameter disks of Fol will be cut and placed on the edge of one half of the Petri plate, adjacent to the center. Plates will then be incubated for 48 hours under supplemental fluorescent lighting at room temperature (Mohammad, 2011). After 48 h incubation, a Trichoderma plug of the same diameter will be added to the opposite side of each plate with Fol (Doilom et al., 2020; Rinu, 2013; Firew, 2016). There will be three replicates of each treatment, and each study will be repeated. Photos of culture growth will be taken at 3, 5, and 7 days. Interactions between Fol and Trichoderma isolates will be evaluated based on radial growth of the pathogen vs. radial growth of Trichoderma (mm) as compared to positive and negative P controls, making notes of zones of inhibition or overlapping between the two colonies, which can be indicated by yellow pigmentation (Mohommad, 2011). 

2: Phosphorus solubilization potential by Trichoderma, as determined by Obj. 1b, will allow for selection of two Trichoderma species for this study with contrasting P solubilization potential, and we expect based on preliminary work, similar biocontrol potential. The experimental design will be a randomized complete block design with five diverse tomato species/genotypes, two Trichoderma isolates and a non-inoculated control, and three P sources (phytate, Al-phosphate, super phosphate) plus a control with no added P. Methods used to cultivate Trichoderma cultures on PDA, create spore suspensions, surface-sterilize seed, planting, and transplanting will follow those of Obj.1a. Pots (10-cm diameter) will be filled with a mixture of 50% sand and 50% field soil with low P availability. The pH of the soil mixture will be recorded at the time of transplanting and the time of harvest. Phosphorus amendments for this study will include a source with organic-P (phytate), a poorly soluble inorganic source (AlPO4), and a highly soluble inorganic source (super phosphate). Data collection (plant height, stem caliper, and biomass) will be as described for Obj. 1a. Leaf tissue P will be determined by digestion and ICP (Water’s Agricultural Lab). TO evaluate root morphology effects, root systems will be washed and analyzed using WinRHIZO. Soil P status will be determined through standard methods (Mehlich 1 extractant). This data along with the average shoot biomass and P content will be used to determine the components of P uptake efficiency based on Lonergan and Asher (1967).

3: Two tomato species/genotypes will be inoculated with one of two Trichoderma with varying P solubilization potential (determined from Obj. 1b & 2) and ideally, similar efficacy for increasing F. oxysporum resistance (Obj. 1a) in a randomized complete block design. Tomato seeds will be treated with one of two Trichoderma isolates and a non-inoculated control. Seeds will be left to establish for 3 weeks before being transplanted into experimental pots with one of two P fertilization rates (none or optimal rate; Al-phosphate). All treatments will be inoculated with Fol, except for a non-inoculated control, with four replicates of each treatment. Plants will be grown within the greenhouse for 9 weeks post transplanting, as described for Obj. 1a and 2. Data collection will be as described in Obj. 2, including disease ratings, measurements of plant height and stem caliper, plant biomass, leaf tissue P concentration, soil P, and root fungal colonization by plating on selective media.