Understanding Trichoderma bio-inoculants in the root ecosystem of Pinus radiata

Oral presentation on understanding Trichoderma bio-inoculants in the root ecosystem of Pinus radiat

Use of Coniothyrium minitans transformed with the hygromycin B resistance gene to study survival and infection of Sclerotinia sclerotiorum sclerotia in soil

A Coniothyrium minitans strain (T3) co-transformed with the genes for β-glucuronidase (uidA) and hygromycin phosphotransferase (hph), the latter providing resistance to the antibiotic hygromycin B, was used to investigate the survival and infection of sclerotia of Sclerotinia sclerotiorum by C. minitans over time in four different soils. Infection of sclerotia was rapid in all cases, with the behaviour of transformant T3 and wild type parent A69 being similar. Differences were seen between the soils in the rate of infection of sclerotia by C. minitans and in their indigenous fungal populations. Amendment of agar with hygromycin B enabled the quantification of C. minitans in soil by dilution plating where there was a high background of other microorganisms. In Lincoln soil from New Zealand, which had a natural but low population of C. minitans the hygromycin B resistance marker allowed the umambiguous discrimination of the applied transformed isolate from the indigenous hygromycin B sensitive one. In this soil, although the indigenous C. minitans population was detected from sclerotia, none were recovered on the dilution plates, indicating the increased sensitivity of C. minitans detection from soil using sclerotial baiting. C. minitans was a very efficient parasite, being able to infect a large proportion of sclerotia within a relatively short time from an initially low soil population. The addition of hygromycin B to agar also allowed the detection of C. minitans from decaying sclerotia by inhibiting secondary fungal colonisers. This is the first report to show that fungi colonising sclerotia already infected by C. minitans mask the detection of C. minitans from sclerotia rather than displacing the original parasite

Grapevines escaping trunk diseases in New Zealand vineyards have a distinct microbiome structure

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome

Botryosphaeriaceae species associated with blueberry dieback and sources of primary inoculum in propagation nurseries in New Zealand

Sampling at blueberry farms found Botryosphaeriaceae fungi in five of seven farms sampled; overall incidence was 41.4%, with Neofusicoccum australe (79.0%), N. luteum (8.0%), N. ribis (8.0%) and N. parvum (5.0%). Sampling of nursery plants found infections in all four nurseries with 45% incidence in mainly asymptomatic plants, which were infected with N. australe (66.0%), N. parvum (31.5%) and N. ribis (2.5%). Asymptomatic propagation cuttings from one nursery were found to have external contamination of Botryosphaeriaceae DNA (90.0%) and internal infection (65.0%) by the four main species found in the blueberry farms and nurseries. For propagation media nested PCR showed that out of the 98 samples received, 43 samples were positive for the presence of Botryosphaeriaceae DNA (44.0%). Results from the SSCP indicated that N. australe, N. luteum, N. parvum/ N. ribis and Diplodia mutila were present in the propagation media received. Isolates of the four main species recovered from farms and nurseries were pathogenic on blueberry stems but pathogenicity differed significantly between species and isolates within a species, with N. ribis being the most pathogenic, then N. parvum, N. luteum and N. australe. Overall, the high rate of infection in nursery plants indicated that nurseries can be a major source of infection for blueberry farms. Since propagating cuttings are the likely sources of infection for nurseries, these should be targeted in the control strategies

Ecological studies of the bio-inoculant Trichoderma hamatum LU592 in the root system of Pinus radiata

The plant health- and growth-promoting biological inoculant (bio-inoculant) Trichoderma hamatum LU592 was transformed with the constitutively expressed green fluorescent protein (gfp) and hygromycin B resistance (hph) genes to specifically monitor the isolate in the root system of Pinus radiata within a strong indigenous Trichoderma population. A modified dilution plating technique was developed to allow the determination of the mycelia proportion of total propagule levels. LU592 was shown to colonize the rhizosphere most effectively when 105 spores per pot were applied compared with inoculum concentrations of 103 and 107 spores per pot. LU592 extended its zone of activity beyond the rhizosphere to at least 1 cm away from the root surface. A positive relationship was shown between P. radiata root maturation and the spatial and temporal proliferation of LU592 in the root system. A steep increase in mycelia levels and proportion of penetrated root segments was observed after 12 weeks. This study reinforces the value of genetic markers for use in ecological studies of filamentous fungi. However, despite isolate-specific recovery of the introduced isolate, it was shown that total propagule counts do not always correlate with the amount of viable mycelium present in the root system. Therefore, it is proposed that the differentiation of mycelia from spores and root penetration is used as more accurate measures of fungal activity

Understanding Trichoderma in the root system of Pinus radiata: associations between rhizosphere colonisation and growth promotion for commercially grown seedlings

Two Trichoderma isolates (T. hamatum LU592 and T. atroviride LU132) were tested for their ability to promote the growth and health of commercially grown Pinus radiata seedlings. The colonisation behaviour of the two isolates was investigated to relate rhizosphere competence and root penetration to subsequent effects on plant performance. Trichoderma hamatum LU592 was shown to enhance several plant health and growth parameters. The isolate significantly reduced seedling mortality by up to 29 %, and promoted the growth of shoots (e.g. height by up to 16 %) and roots (e.g. dry weight by up to 31 %). The introduction of LU592 as either seed coat or spray application equally improved seedling health and growth demonstrating the suitability of both application methods for pine nursery situations. However, clear differences in rhizosphere colonisation and root penetration between the two application methods highlighted the need for more research on the impact of inoculum densities. When spray-applied, LU592 was found to be the predominant Trichoderma strain in the plant root system, including bulk potting mix, rhizosphere and endorhizosphere. In contrast, T. atroviride LU132 was shown to colonise the root system poorly, and no biological impact on P. radiata seedlings was detected. This is the first report to demonstrate rhizosphere competence as a useful indicator for determining Trichoderma bio-inoculants for P. radiata. High indigenous Trichoderma populations with similar population dynamics to the introduced strains revealed the limitations of the dilution plating technique, but this constraint was alleviated to some extent by the use of techniques for morphological and molecular identification of the introduced isolates

Ecological studies of Trichoderma bio-inoculants in the soil ecosystem of Pinus radiata

Oral presentation on ecological studies of Trichoderma bio-inoculants in the soil ecosystem of Pinus radiata

Fate of mycelial and conidial propagules of Ilyonectria and Dactylonectria species in soil

Black foot disease of grapevines causes significant economic loss to the viticulture industry worldwide. A novel method was developed to investigate the fate of propagules of three species associated with black foot disease in New Zealand, Dactylonectria macrodidyma, Ilyonectria europaea and I. liriodendri, in soil. Conidia or mycelium of one isolate each of the three species were buried in soil in nylon mesh bags, and conidia/chlamydospore numbers were determined microscopically after 2 and 3 weeks. Conidia and chlamydospores were produced by mycelial inocula of all isolates, with greater numbers of chlamydospores after 3 weeks. Conidial inocula of all isolates also produced chlamydospores. Chlamydospores were formed at either the terminus or side of a hypha, and single and multiple conidia formed chlamydospores by combining their cellular protoplasm. Chlamydospores were produced from conidia, and conidia from mycelium faster for the I. europaea isolate than the D. macrodidyma and I. liriodendri isolates. The rapid formation of chlamydospores as survival propagules will facilitate the ability of these pathogens to persist in soil in the absence of a host