Endophytic fungal isolates from apple tissue: Latent pathogens lurking within?

Fungal endophytes inhabit a similar ecological niche to that occupied by many phytopathogens, with several pathogens isolated from healthy tissues in their latent phase. This study aimed to evaluate the pathogenicity, the colonisation ability, and the enzyme activity of 37 endophytic fungal isolates recovered from apparently healthy apple shoot and leaf tissues. The pathogenicity of the isolates was assessed on 'Royal Gala' and 'Braeburn' fruit and detached 'Royal Gala' shoots. For the non-pathogenic isolates, their ability to endophytically colonise detached 'Royal Gala' shoots was evaluated. Enzyme activity assays were undertaken to determine whether the pathogenicity of the endophytes was related to the production of the extracellular enzymes, amylase, cellulase, pectinase, protease, and xylanase. Of the 37 isolates studied, eight isolates, representing the genera Colletotrichum, Diaporthe, Fusarium, and Penicillium, were shown to be pathogenic on both apple shoots and fruit. Two isolates identified as Trichoderma atroviride, were pathogenic only on shoots, and three isolates, representing the genus Diaporthe, were pathogenic only on fruit. Of the remaining 24 isolates, 22 (Biscogniauxia (n = 8), Chaetomium (n = 4), Trichoderma (n = 3), Epicoccum (n = 2), Neosetophoma (n = 2), Xylaria (n = 1), Daldinia (n = 1), and Paraphaeosphaeria (n = 1)) were recovered from the inoculated apple shoots but two failed to colonise the shoot tissues. Of the isolates tested, 20 produced amylase, 15 cellulase, 25 pectinase, 26 protease, and 13 xylanase. There was no correlation between the range and type of enzymes produced by the isolates and their pathogenicity or ability to endophytically colonise the shoot tissue. The study showed that approximately one-third (13/37) of the isolates recovered from the apparently healthy apple shoot tissues were observed as latent pathogens. The isolates that did not cause disease symptoms may have the ability to reduce colonisation of apple tissues by pathogens including Neonectria ditissima associated with European canker of apple.

In vitro inhibition of Sclerotinia sclerotiorum mycelial growth and reduction of sclerotial viability by the volatile bioactive compounds of Brassicaceae crops.

AIMS: Sclerotinia sclerotiorum is an important pathogen of a wide range of crops, with current control mostly relying on the use of fungicides. This study assessed the effect of biofumigation on in vitro inhibition of mycelial growth and reduction of sclerotial viability of S. sclerotiorum as an attempt to seek an alternative management strategy. METHODS AND RESULTS: The effect of different biofumigant crop types to inhibit mycelial growth of ten S. sclerotiorum isolates was investigated, with Brassica juncea 'Caliente 199' being the most effective biofumigant crop. The efficacy of 'Caliente 199' to inhibit mycelial growth and reduce sclerotial viability was influenced by different crop factors. Plant tissue of 'Caliente 199' harvested at 50% or 100% flowering and adjusted to 80% (w/w) moisture resulted in greater mycelial inhibition and a reduction in the sclerotial viability compared with the vegetative tissue with the same plant moisture. Mycelial inhibition and reduction of sclerotial viability were affected by tissue quantity. Whole plant tissue and shoots only resulted in a similar inhibition of mycelial growth, but whole plant tissue resulted in a greater reduction of sclerotial viability. The S. sclerotiorum isolates differed in sensitivity to the volatile bioactive compounds released by the biofumigant plant tissue. CONCLUSIONS: The volatile bioactive compounds released by 'Caliente 199' resulted in effective mycelial inhibition but did not kill sclerotia completely.

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.

Soil Arbuscular Mycorrhizal Fungal Communities Differentially Affect Growth and Nutrient Uptake by Grapevine Rootstocks.

Arbuscular mycorrhizal fungi (AMF) deliver potentially significant services in sustainable agricultural ecosystems, yet we still lack evidence showing how AMF abundance and/or community composition can benefit crops. In this study, we manipulated AMF communities in grapevine rootstock and measured plant growth and physiological responses. Glasshouse experiments were set up to determine the interaction between rootstock variety and different AMF communities, using AMF communities originating under their own (i.e., "home") soil and other rootstocks' (i.e., "away") soil. The results revealed that specific AMF communities had differential effects on grapevine rootstock growth and nutrient uptake. It was demonstrated that a rootstock generally performed better in the presence of its own AMF community. This study also showed that AMF spore diversity and the relative abundance of certain species is an important factor as, when present in equal abundance, competition between species was indicated to occur, resulting in a reduction in the positive growth outcomes. Moreover, there was a significant difference between the communities with some AMF communities increasing plant growth and nutrient uptake compared with others. The outcomes also demonstrated that some AMF communities indirectly influenced the chlorophyll content in grapevine leaves through the increase of specific nutrients such as K, Mn, and Zn. The findings also indicated that some AMF species may deliver particular benefits to grapevine plants. This work has provided an improved understanding of community level AMF-grapevine interaction and delivered an increased knowledge of the ecosystem services they provide which will benefit the wine growers and the viticulture industry.

Diversity and Bioactivity of Endophytic Actinobacteria Associated with Grapevines.

Grapevine trunk diseases (GTDs) are a significant problem for New Zealand viticulture. Endophytic actinobacteria are of interest as potential biocontrol agents due to their ability to inhibit plant pathogens and improve plant growth. However, no studies have investigated the diversity of actinobacteria associated with grapevines in New Zealand vineyards and their bioactivity. Actinobacteria diversity in different 'Sauvignon blanc' vine tissues from three vineyards (conventional and organic management, and different vine ages) was assessed using different methods and media. Forty-six endophytic actinobacteria were isolated, with more isolates recovered from roots (n = 45) than leaves (n = 1) and shoot internodes (n = 0). More isolates were recovered from the organic (n = 21) than conventional (n = 8) vineyard, mature (25-year old; n = 21) than young (2-year old; n = 2) vines and using a tissue maceration technique (n = 40). Actinomycete Isolation Agar, International Streptomyces Project 2, and Starch Casein media were effective for actinobacteria isolation. Most of the isolates recovered belonged to Streptomyces, with one isolate identified as Mycolicibacterium. Forty isolates were assessed for antifungal activity and plant growth-promoting (PGP) characteristics. Of these, 13 isolates had antifungal activity against test GTD pathogens (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum, and N. luteum). Eighteen isolates exhibited more than one PGP trait; 25siderophore production (n = 25), phosphate solubilization (n = 6), and indole acetic acid production (n = 16). Two strains, Streptomyces sp. LUVPK-22 and Streptomyces sp. LUVPK-30, exhibited the best antifungal and PGP properties. This study revealed the diversity of culturable endophytic actinobacteria from grapevines in New Zealand vineyards and their biocontrol potential against GTD pathogens.

Quantitative genetic analysis reveals potential to breed for improved white clover growth in symbiosis with nitrogen-fixing Rhizobium bacteria.

White clover (Trifolium repens) is integral to mixed pastures in New Zealand and temperate agriculture globally. It provides quality feed and a sustainable source of plant-available nitrogen (N) via N-fixation through symbiosis with soil-dwelling Rhizobium bacteria. Improvement of N-fixation in white clover is a route to enhancing sustainability of temperate pasture production. Focussing on seedling growth critical for crop establishment and performance, a population of 120 half-sibling white clover families was assessed with either N-supplementation or N-fixation via inoculation with a commercial Rhizobium strain (TA1). Quantitative genetic analysis identified significant (p < 0.05) family additive genetic variance for Shoot and Root Dry Matter (DM) and Symbiotic Potential (SP), and Root to Shoot ratio. Estimated narrow-sense heritabilities for above-ground symbiotic traits were moderate (0.24-0.33), and the strong (r ≥ 0.97) genetic correlation between Shoot and Root DM indicated strong pleiotropy or close linkage. The moderate (r = 0.47) phenotypic correlation between Shoot DM under symbiosis vs. under N-supplementation suggested plant growth with mineral-N was not a strong predictor of symbiotic performance. At 5% among-family selection pressure, predicted genetic gains per selection cycle of 19 and 17% for symbiotic traits Shoot DM and Shoot SP, respectively, highlighted opportunities for improved early seedling establishment and growth under symbiosis. Single and multi-trait selection methods, including a Smith-Hazel index focussing on an ideotype of high Shoot DM and Shoot SP, showed commonality of top-ranked families among traits. This study provides a platform for proof-of-concept crosses to breed for enhanced seedling growth under Rhizobium symbiosis and is informative for other legume crops.

AMF Community Diversity Promotes Grapevine Growth Parameters under High Black Foot Disease Pressure.

Black foot disease is one of the main grapevine root diseases observed worldwide and is especially problematic in New Zealand. Arbuscular mycorrhizal fungi (AMF) have been shown to reduce infection and mitigate the effect of black foot disease on grapevine rootstocks. In contrast to prior studies, which have limited their focus to the effect of one, two or a combination of only a small number of AMF species, this study used whole AMF communities identified from 101-14, 5C and Schwarzmann rootstocks sampled from New Zealand vineyards. The effect of AMF on black foot disease was investigated in a 'home' and 'away' experiment using three commercial grapevine rootstocks. The study produced some evidence that AMF treatments lowered disease incidence at 5 cm and disease severity in vines by 40% to 50% compared to the vines inoculated with the pathogen only. This work also showed that the presence of high disease incidence may have limited the potential disease protective effect of AMF community. However, despite the high disease incidence and severity, AMF inoculation increased vine growth parameters by 60% to 80% compared to the vines inoculated with the pathogen only. This study is the first to provide an understanding on how young grapevine rootstocks inoculated with their 'home' and 'away' AMF communities would respond to challenge with a black foot pathogen species mixture. Further research is required to understand the mechanistic effect of AMF colonization on the increase of grapevine growth parameters under high black foot disease pressure.

Influence of blueberry tissue type, wounding and cultivar on susceptibility to infection by Neofusicoccum species.

AIM: Botryosphaeriaceae causing stem blight and dieback of blueberry are important pathogens limiting economic production worldwide. This study investigated the pathogenicity and relative virulence of isolates from the Neofusicoccum species commonly associated with blueberries in New Zealand on different tissues and cultivars of blueberries. METHODS AND RESULTS: Both wounded and non-wounded fruit and flower buds and wounded attached soft green and hard green shoots were susceptible to infection by conidia of Neofusicoccum australe, Neofusicoccum parvum and Neofusicoccum ribis. N. ribis was generally most virulent, followed by N. parvum and then N. australe. Inoculation of potting mixture with N. australe or N. ribis conidia showed that potting mixtures were not a source of inoculum for infection of blueberry roots. Wounded and non-wounded leaf buds, fruit and wounded soft green shoots and hard green shoots of the different cultivars tested were susceptible to infection by N. parvum and N. ribis. Whilst the fruit of all cultivars were similarly infected, infection incidence in inoculated leaf buds was lowest in "Blue Bayou" and "Ocean Blue". Cultivar susceptibility differed when tested on soft green shoots compared with hard green shoots, with shortest lesions developed on "Maru" on soft green shoots, and "Centra Blue" and "Ocean Blue" on hard green shoots. CONCLUSIONS: All tested above-ground blueberry tissues, including non-wounded tissue, were susceptible to Neofusicoccum spp. All the cultivars assessed were susceptible to infection, although they varied in their relative susceptibility depending on the tissue assessed. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for non-wounded tissue to become infected indicate that fungicides may need to be applied to protect all tissue, not just wounds.

Genetic mapping of a barley leaf rust resistance gene Rph26 introgressed from Hordeum bulbosum.

KEY MESSAGE: The quantitative barley leaf rust resistance gene, Rph26, was fine mapped within a H. bulbosum introgression on barley chromosome 1HL. This provides the tools for pyramiding with other resistance genes. A novel quantitative resistance gene, Rph26, effective against barley leaf rust (Puccinia hordei) was introgressed from Hordeum bulbosum into the barley (Hordeum vulgare) cultivar 'Emir'. The effect of Rph26 was to reduce the observed symptoms of leaf rust infection (uredinium number and infection type). In addition, this resistance also increased the fungal latency period and reduced the fungal biomass within infected leaves. The resulting introgression line 200A12, containing Rph26, was backcrossed to its barley parental cultivar 'Emir' to create an F2 population focused on detecting interspecific recombination within the introgressed segment. A total of 1368 individuals from this F2 population were genotyped with flanking markers at either end of the 1HL introgression, resulting in the identification of 19 genotypes, which had undergone interspecific recombination within the original introgression. F3 seeds that were homozygous for the introgressions of reduced size were selected from each F2 recombinant and were used for subsequent genotyping and phenotyping. Rph26 was genetically mapped to the proximal end of the introgressed segment located at the distal end of chromosome 1HL. Molecular markers closely linked to Rph26 were identified and will enable this disease resistance gene to be combined with other sources of quantitative resistance to maximize the effectiveness and durability of leaf rust resistance in barley breeding. Heterozygous genotypes containing a single copy of Rph26 had an intermediate phenotype when compared with the homozygous resistant and susceptible genotypes, indicating an incompletely dominant inheritance.

The Bacterial Signature of Leptospermum scoparium (Manuka) Reveals Core and Accessory Communities with Bioactive Properties.

Leptospermum scoparium or manuka is a New Zealand native medicinal plant that produces an essential oil with antimicrobial properties. This is the first study to investigate the structure and bioactivity of endophytic bacteria in manuka by using a combination of cultivation-independent (DGGE) and dependent approaches. A total of 23 plants were sampled across three sites. Plants were considered either immature (3-8 years) or mature (>20 years). The endophyte community structure and richness was affected by plant tissue and bacterial communities became more stable and uniform as plant maturity increased. A total of 192 culturable bacteria were recovered from leaves, stems and roots. Some bacterial isolates showed in vitro biocontrol activity against two fungal pathogens, Ilyonectria liriodendri and Neofusicoccum luteum and a bacterial pathogen, Pseudomonas syringae pv. actinidiae. A high proportion of bacterial endophytes could produce siderophores and solubilise phosphate in vitro. Gammaproteobacteria was the most variable class, representing the majority of cultivated bacteria with bioactivity.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards.

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.

Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia.

Water availability is an important environmental factor which has major effects on fungal activity. The effects of osmotic (KCl amended agar) and matric Polyethylene glycol ((PEG) 8000 amended agar) potentials over the range -0.1 to -5.0MPa on mycelial growth and conidial germination of eight isolates of the sclerotial parasite Coniothyrium minitans was assessed. The influence of soil water potential on the ability of three selected isolates (LU112, LU545, and T5R42i) to parasitise sclerotia of the plant pathogen Sclerotinia sclerotiorum was determined. For all eight C. minitans isolates, decreasing osmotic and matric potentials caused a reduction in mycelial growth and conidial germination. Isolates were more sensitive to decreasing matric potential than osmotic potential. Across the isolates, growth at an osmotic potential of -5.0MPa was 30-70% of the growth seen in the control, whereas less than 20% of the control growth was seen at the corresponding matric potential. Across all isolates no conidial germination was seen at matric potential of -5.0MPa. The C. minitans isolates varied in their sensitivity to decreasing water potentials. Mycelial growth and conidial germination of three isolates (LU112, Conio, and CH1) were more tolerant of low osmotic potential and matric potential with respect to mycelial growth. Isolates T5R42i and LU430 were least tolerant. In contrast, conidial germination of isolates Conio, LU545, and T5R42i were less sensitive to decreasing matric potential. Soil water potential was seen to affect infection and viability of sclerotia by the three C. minitans isolates. Isolate LU545 reduced sclerotial viability over a wider water potential range (-0.01 to -1.5MPa) compared with LU112 (-0.01 to -1.0MPa), with isolate T5R42i being intermediate. Indigenous soil fungi (Trichoderma spp. and Clonostachys rosea) were recovered from sclerotia but did not result in reduction in sclerotial viability. The relevance of these results in relation to biocontrol activity of C. minitans in soil is discussed.

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.

Ectomycorrhizal species associated with Pinus radiata in New Zealand including novel associations determined by molecular analysis.

Ectomycorrhizal (ECM) associates of the exotic plantation species Pinus radiata were investigated above and below ground over two years in the North Island of New Zealand. ECM species were identified using morphological and molecular (restriction fragment length polymorphism and DNA sequencing) analysis. Eighteen ECM species were observed fruiting above ground; 19 ECM species were identified below ground. In the above ground study, Wilcoxina mikolae, Rhizopogon pseudoroseolus and Inocybe sindonia were noted for the first time as ECM associates of P. radiata in New Zealand. Below ground, the species W. mikolae, R. pseudoroseolus, Rhizopogon luteorubescens, Pseudotomentella sp., Pseudotomentella tristis and Tomentella sp. were found as new associates of P. radiata in New Zealand. Additionally, six ECM types were found that could not be identified with molecular analysis. The putative ECM taxa Tricholoma pessundatum, Laccaria laccata and Hebeloma crustuliniforme were examined by molecular analysis, and species identifications were proposed to be changed to Tricholoma sp., L. laccata and Hebeloma sp. for specimens associated with P. radiata in New Zealand. The species identity of I. sindonia, previously unidentified to species level, was determined with direct sequencing.

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 beta-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.