In 2023, vetch plants with wilting symptoms (root and crown discolouration) were observed in the Esperance region, Western Australia (WA), prompting an investigation into the underlying causes. Wilted plant parts incubated in moist trays revealed associated Fusarium species. The fungus was isolated from infected root and stem tissue by surface sterilising with 10% sodium hypochlorite solution for one minute and rinsing three times with sterilised water before plating on potato dextrose agar (PDA) medium containing streptomycin and ampicillin. The plates were incubated at 27°C, under a 12-hour day/night cycle for one week. Hyphal tips from colony margins were transferred onto PDA with antibiotics to obtain pure cultures. The identity of the fungus was confirmed by the Department of Primary Industries and Regional Development (DPIRD) Diagnostic Laboratory Services (DDLS) in WA as Fusarium acuminatum (Spec ID; 0001b, 0001C, 0002A, 0003, 0004) by sequencing the ITS, LSU, TEF-1α and RPB2 gene regions.
The isolated F. acuminatum was used for pathogenicity study using a soil inoculation method.
A cornmeal sand mixture was prepared by mixing 240g of sandy soil (pH neutral) with 26g of cornmeal and adding 65ml of distilled water to 500mL Erlenmeyer flasks. The mixture was autoclaved twice at 121°C for 30min. The cornmeal sand inoculum (CSI) was prepared by adding PDA disks (5mm diameter) with and without the Fusarium fungal mycelium and incubating at 27°C for 4 weeks without shaking. The CSI was then mixed with sterilised soil at a volume ratio of 3:7 and used to fill pots. Two hundred millilitres of this mixture was used as potting mix for each experimental pot (72 x 72 x 100 mm) for planting the pre-germinated vetch seeds.
Inoculation experiments were conducted in a glasshouse at 20°C day / night temperature and frequent hand watering with sterilised deionised water. The experiment comprised three replicates of three treatments. The treatments were: 1) pre-germinated vetch seeds (cv Timok) planted into cornmeal sand inoculum (CSI) without fungal mycelium (control), 2) pre-germinated vetch seeds planted into CSI with fungal mycelium (single inoculation), and 3) pre-germinated vetch seeds transferred to Fusarium acuminatum PDA plates for a week before planting into CSI with fungal mycelium (double inoculation).
Five pre-germinated seedings were planted in each pot, with all pots being kept in the glasshouse and plants monitored regularly for wilting symptoms. Four weeks after planting, the double inoculated treatment had higher proportion of wilted plants (p=0.005, chi-squared test) and lower plant height (p=0.074 based on ANOVA on pot average data or p=0.010 based on t-test of individual plant heights) compared to the control. The results for the single inoculation treatment were approximately midway between the control and double inoculation treatments and not significantly different from either. F. acuminatum. was recovered from wilted seedlings fulfilling the requirements for Koch’s postulate.
This study provides compelling evidence that F. acuminatum was the causative agent of root rot and wilting affecting vetch. Furthermore, it is the first report of the presence of this pathogen on vetch in Australia.