An investigation into the potential mycoherbicidal properties of four fungal isolates for use in the control of giant buttercup ( Ranunculus acris L.): A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Agricultural Science (Honours)

Abstract

Four fungal isolates (two possible Gnomonia spp GBX,CBY, one Fusarium spp and one Alternaria spp) were tested as prospective mycoherbicides against Ranunculus acris L. The techniques that were used included, pathogenicity testing on whole plants and excised tissue, scanning electron microscopy to show the characteristics of the leaf surface and spore germination and a method of leaf clearing and staining to show the progression of the possible Gnomonia spp through the leaf tissue. The plant pathogenicity testing showed that GBX was the most pathogenic isolate on both excised and whole plant tissue, followed by CBY and the Fusarium spp. Alternaria was found to inhibit GBX to such an extent that its pathogenicity was almost completely removed on excised tissue. The isolates GBX and CBY were able to kill excised leaf tissue within 80 h. The results showed that GBX lesion growth was significantly greater (P=0.05) than all other isolates when it was inoculated onto excised petioles and flowering stems. The lesions covered the 80 mm of the excised tissue portions in 112 h when the tissue was inoculated with GBX distally, followed by CBY and the GBX/Alternaria mix at 132 h with the Fusarium isolate failing to reach the end of the 80mm piece of excised flowering stem or petiole. The results for proximally inoculated excised tissue showed that the Fusarium isolate was the most pathogenic but progress of the lesion down the excised petiole and flowering stems was a lot slower. Lesions of the Fusarium isolate took 172 h when applied proximally to flowering stem to reach 76 mm and 166 h to reach 75 mm in length on the proximally inoculated excised petiole tissue. The isolate GBX had the second longest lesions of 67mm on the flowering stem and 68mm on the petiole. These lesion lengths were significantly (P=0.05) different from the CBY lesion length. All fungal isolates were unable to produce symptoms of infection on excised root and crown tissue, which was considered a highly significant observation. Whole plant inoculations were difficult to achieve with only 40% of the GBX inoculated plants, and 30% of the CBY inoculated plants developing lesions after inoculation. Although GBX and CBY caused the death of a large majority of leaves (70-80% after eight days), R. acris was able to recover quickly and started to produce new leaves 10 days after inoculation. The leaf death was caused by two methods of action: 1) the pathogen infected the leaf and destroyed the leaf tissue, or 2) the pathogen grew on the petioles and caused lesions that encircled the petiole causing it to break at the base. Young expanding leaves on whole plants inoculated with GBX and CBY, were only marginally affected by the pathogens, and were not killed. The pathogenicity testing came up with seven key observations which were 1) crowns and roots of R. acris were not infected by any of the fungal pathogens, 2) GBX and CBY lesions grew more quickly on whole plants of R.acris when visually compared to lesion growth on excised tissue, 3) GBX was unstable in culture, and sectored after two subcultures, 4) GBX and CBY were the most pathogenic isolates examined 5) Alternaria inhibited GBX in the GBX/Alternaria spore suspension, 6) there was some difficulty in inoculating whole R .acris plants, 7) growth of the fungi basipetally was faster on excised petiole and flowering stem tissue than acropetal development. Scanning electron microscopy showed that the leaves had a relatively smooth surface with no ornamental wax projections. The stomata were relatively large (40-50µm by 20-26µm) with more stomata occurring per unit area on the abaxial leaf surface (350-380/cm²) compared to the adaxial surface (250-300/cm²). The spores of GBX and CBY were relatively similar in size, 12-16µm in length by 2-2.5 µm in diameter and consisted of two cells joined in middle with a collar. The Fusarium isolate had four cells and was up to 35µm in length by 2.5-4.5 µm in diameter. The Fusarium isolate germinated in 6-12 hours but did not produce appressorium. The isolate GBX was the quickest to germinate in 2 h and had formed appressorium by 12 h after inoculation of plant material. The isolate CBY germinated within 4 h but no appressorium were observed 12 h after inoculation. At this time germtubes in GBX and CBY had reached up to 50 µm in length. The method of leaf clearing and staining showed that hyphal growth preceded lesion growth. This showed up as blue black hyphae against the pale blue background of host tissue. The overall conclusion is that considerably more research would be required to determine the feasibility of developing the most promising isolates GBX and CBY as potential mycoherbicides for the control of R.acris.