G. Burch

Growth patterns of bacterial communities in the rhizoplane and rhizosphere of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) in long-term pasture

Rhizoplane bacteria in white clover roots (0–5 cm), perennial ryegrass roots (0–5 and 5–10 cm) and surrounding rhizosphere soil from a long-term pasture were studied for the rate of colony development over a 10 day period. Serial dilutions were inoculated onto 0.1 strength tryptose soy agar (TSA) and 0.1 strength modified Kings medium B (KB). Bacterial counts were made eight times over 10 days. Bacterial colony development (CD) and eco-physiological (EP) indices were calculated from these data. There were no significant differences in CD and EP indices of fluorescent pseudomonads growing on KB medium. After 5 days, the number of colonies developing on KB as a proportion of total colonies appearing over 10 days was lower for ryegrass roots than for soil (P < 0.05). The community structure derived from the colonies growing on 0.1 strength TSA showed that the rhizoplanes of white clover and ryegrass had a greater proportion of K-strategists and a smaller proportion of r-strategists than did the rhizosphere soil (P < 0.001). CD values for roots were significantly lower than those for soil (P < 0.001), while EP values were not significantly different. Randomly selected bacterial isolates were identified using the Microlog™ system. The fastest growing (24 h) strains belonged to the genera Pseudomonas and Bacillus spp. Other genera identified included Acinetobacter, Sphingomonas, Streptococcus, Cellulomonas, Comamonas, Xanthomonas and Erwinia. The slowest growing bacteria belonged to the genera Cellulomonas, Rhizobium, Xanthomonas and Erwinia.

Effects of chitin amendment of soil on microorganisms, nematodes, and growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.)

Effects of soil amendment with crabshell chitin on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.), and on populations of soil bacteria, fungi, and plant-parasitic and free-living nematodes were investigated in a pot trial. Five soil samples were collected from Te Puke (Paengaroa Shallow Sand, a Typic Hapludand) and five from Hamilton (Bruntwood silt loam, an Aquic Hapludand), New Zealand. Subsamples of each soil were either amended with chitin or unamended and planted with white clover and ryegrass. The ryegrass shoot weight in amended soil was greater (P<0.01), most probably due to N mineralised from chitin. A significantly lower (P<0.01) root: shoot ratio of ryegrass in the amended soil also suggested improved N availability, and therefore less root mass was needed to support a given shoot mass. A reduction in nodulation was observed in 12-day-old white clover seedlings (P<0.05) and also in 6-week-old seedlings (P<0.01). The shoot weight of white clover was significantly lower (P<0.05) in amended soil, possibly due to phytotoxic effects of chitin. Chitin increased (P<0.01) the populations of bacteria and fungi by 13-fold and 2.5-fold, respectively. The cyst nematode of white clover, Heterodera trifolii, was significantly reduced in chitin-amended soil, possibly due to increased levels of chitinase produced by rhizosphere microorganisms. Two other plant-parasitic nematodes, Pratylenchus spp. and Tylenchus spp., were also reduced in ryegrass roots and in soil as a result of the chitin amendment. However, the total number of free-living nematodes increased 5.4-fold in amended soil.

Effects of plant-parasitic nematodes and rhizosphere microorganisms on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.)

A pot trial was carried out to study the effects of plant-parasitic nematodes and rhizosphere microorganisms on the growth of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). Plants were grown together in Horotiu sandy loam (Vitric Hapludand). The treatments consisted of: untreated field soil (UT); soil frozen to −20°C to reduce nematodes (FR); soil fumigated with chloroform to kill most soil organisms (FU); and fumigated soil inoculated with a layer of frozen soil to reintroduce organisms present in frozen soil (FUI). The mean wet weights of white clover and ryegrass grown in UT soil for 6–9 weeks were only 10 and 60%, respectively, of those grown in FR soil and 5.8 and 56%, respectively, of those grown in FU soil. The severe growth reduction of white clover in UT was attributed to early invasion of roots by two nematode species, Heterodera trifolii and Meloidogyne hapla, which were detected in stunted 7-day-old seedlings. In the FR and FUI treatments, the freeze-thaw process appeared to have killed the second stage juveniles but not the eggs, which hatched to release more juveniles to invade white clover roots. Plant growth in FR, and to a lesser extent in FUI treatments, was reduced in comparison with FU, where there was no nematode invasion. The bacterial numbers in surface-sterilized roots were unaffected by any of the treatments. This suggests that the entry of bacteria into root tissues is independent of the wounding caused by nematodes. The mean bacterial numbers of the medians (means of log cfus across treatments and plant species of the median numbers across replicates) for “total” bacteria, fluorescent pseudomonads, Gram-negative bacteria and Gram-positive bacteria from surface-sterilized roots were 4.26, < 2, 2.94 and 3.08 for the four treatments, respectively. The most common bacterial genera identified were Pseudomonas and Bacillus. Fungi isolated from surface-sterilized roots included Fusarium oxysporum, Codinaea fertilis and many sterile fungi, with F. oxysporum being the most common identified fungus in FU and FUI treatments. C. fertilis was killed by the soil freezing process. F. oxysporum did not appear to be associated with a decrease in dry matter production of either white clover or ryegrass, but C. fertilis may have adversely affected the ryegrass dry matter production in the treatment UT.

Biodiversity of indigenous tussock grassland sites in Otago, Canterbury and the central North Island of New Zealand III. Soil microorganisms

Abstract Bacterial and fungal communities in indigenous tussock grassland soils were studied at four locations, Mt Benger and Deep Stream (Otago), Cass (Canterbury) and Tukino (central North Island). Soil samples collected from inter‐tussock and tussock areas were used to enumerate total culturable bacteria (colony forming units (CFU)) as well as a number of specific groups of bacteria, fungal types and diversity, and soil microbial functional diversity. Soil microbial biomass carbon (C) and nitrogen (N) as well as extractable C and N were also determined. Fungal populations were lowest at Mt Benger while fluorescent Pseudomonas was lowest at Cass. In these indigenous soils, bacterial CFU were c. 30 times lower and fungal populations c. 10 times greater than in developed New Zealand pastoral soils. CFU of bacteria in complex media (r‐strategists) were similar in magnitude to bacteria growing on weak CA medium (K‐strategists). The microbial biomass C in indigenous grassland soils were also significantly greater than that found in developed pastoral soils, possibly due to dominance of fungi in indigenous soils. Microbial C and N were lower in Cass and Tukino sites compared to Mt Benger and Deep Stream sites. Microbial nitrogen was also significantly greater (P < 0.001) in inter‐tussock than under tussock samples. The functional diversity of soil microbes, was significantly greater (P< 0.01) in inter‐tussock samples compared to tussock samples at all sites except Mt Benger. The total microbial activity, as measured by the colour development in Biolog plates (Average Well Colour Development (AWCD)), was low in Cass soil compared to others. Eighty‐three types of fungi belonging to 30 genera were recovered from the four sites. The diversity of fungi found in Otago sites was greater than in the other two sites (P < 0.001). Fusarium spp. that are often common in pastoral soils were conspicuous by their rarity in indigenous soils.

Microbial pathogens and plant parasitic nematodes in pastures with declining vigour.

Abstract Two years after establishment, areas of low vigour (LV) were noticed within a white clover (Trifolium repens)/ryegrass (Lolium perenne) pasture. These areas became progressively larger, and, two years later, the pasture had low vigour, comprising only 3% white clover. Clover tissue nitrogen and phosphorus levels were greater in high vigour (HV) than in LV herbage. There were no differences (P > 0.05) in “total” bacteria, fluorescent pseudomonads, or proportions of deleterious bacteria in LV and HV white clover roots (mean logio CFUs for total count and fluorescent pseudomonads were 9.4 and 8.8, respectively, and the proportions of deleterious bacteria encountered in these groups were 6.9% and 17.5%). There was a 9‐fold difference (P < 0.01) in the white clover dry matter between LV areas and HV areas in February, increasing to 25‐fold (P< 0.01) in March. The total dry matter yield differences, which included grasses and clover, were 2.7‐ and 2‐fold (P < 0.01) for the two months. More fungi were isolated (P < 0.05) from LV white clover roots than from HV roots (164 and 104 isolates, respectively). This was mostly due to greater numbers (P < 0.01) of Codinaea fertilis in LV roots (111 cf. 41 in HV). In ryegrass roots, there were also greater numbers (P < 0.01) of total fungi in LV roots (97 cf. 50 total isolates in HV), mostly due to higher populations of Fusarium oxysporum in LV roots (29 isolates cf. 5 in HV). There was no difference (P > 0.05) in the Soil Pathogenicity Index between LV and HV soil. The plant parasitic nematode populations in 250 ml of soil in LV and HV areas were 42.7 and 9.7 for Heterodera and 28.1 and 3.9 for Meloidogyne, respectively.

Detection of Invertebrate Suppressive Soils, and Identification of a Possible Biological Control Agent for Meloidogyne Nematodes Using High Resolution Rhizosphere Microbial Community Analysis

White clover (Trifolium repens) is the key legume component of New Zealand pastoral agriculture due to the high quality feed and nitrogen inputs it provides. Invertebrate pests constrain white clover growth and this study investigated rhizosphere-associated fungal controls for two of these pests and attempts to disentangle the underpinning mechanisms. The degree of suppressiveness of 10 soils, in a latitudinal gradient down New Zealand, to added Meloidogyne hapla and Costelytra zealandica scarab larvae was measured in untreated soil. Most of the soils showed no suppressive activity against these pests but two showed activity against M. hapla and two against C. zealandica. Rhizosphere fungi responsible for pest suppressive responses were elucidated via next-generation sequencing. In the M. hapla-suppressive soils nematode-trapping Orbiliomycetes fungi were present in significantly greater abundance than non-suppressive soils and their abundance increased further with addition of M. hapla. A comparison of plant growth and the rhizosphere fungal community between untreated and irradiated soil was carried out on 5 of the 10 soils using Pyronota as the scarab larvae. Soil irradiation either: reduced (by 60–70%); increased (16×) or made no difference to white clover growth across the five soils tested, illustrating the range of microbial impacts on plant production. In one of the M. hapla suppressive soils irradiation resulted in a significant increase in nematode galling suggesting that Orbiliomycetes fungi were indeed responsible for the suppressive effect. Lack of consistent changes in soil macronutrients and pH post-irradiation suggest these were not responsible for plant or invertebrate responses. The use of next generation sequencing in controlled pot trials has allowed identification of a potential biological control organism and bioindicator for M. hapla suppression.

Toxic and antibiotic properties of red thread, Laetisaria fuciformis

An isolate ofLaetisaria fuciformis in axenic culture did not produce toxic metabolites in concentrations sufficient to affect rats or sheep or mammalian cells in tissue culture, nor did it produce ergot alkaloids, paxilline or zearalenone in amounts detectable by ELISA. It did produce diffusable compound(s) inhibitory to a range of Gram-positive and-negative bacteria but not to the mould species tested.

An improved bioassay for screening bacteria for biocontrol activity against slugs that avoids volatile-induced slug mortality

Abstract A strain of Stenotrophomonas maltophila previously thought to have molluscicidal activity was tested using a standard bioassay. Initial results on host range and dose response were promising. However, slug mortality was induced by volatile compounds released from the decaying food rather than by infection. We propose a modification of the bioassay that avoids this problem.