Fungal endophytes from salt-adapted plants confer salt tolerance and promote growth in wheat (Triticum aestivum L.) at early seedling stage.

With increasing human global population, increased yield under saline conditions is a desirable trait for major food crops. Use of endophytes, isolated from halophytic hosts, seems to be an exciting approach for conferring salt tolerance to a salt-sensitive crop. Therefore, in the current study, fungal endophytes were isolated from halophytic plants' roots and their ability to withstand in vitro salt stress was evaluated. The fungal endophytes could withstand up to 1M NaCl concentrations and this tolerance was independent of their host or tissue source. When inoculated on salt-sensitive wheat seeds/seedlings, several of the endophytes showed a positive impact on germination and biomass-related parameters upon salt stress, both in vitro and under glasshouse conditions. One of the isolates from dicot plants (identified as Microsphaeropsis arundinis) could successfully colonize wheat and promote its growth under salt and no-salt conditions. Amongst the fungal isolates that are known to be natural endophytes of wheat, Chaetomium globosum was the best performing isolate and has previously been reported to be an effective biocontrol agent. Based on the results of our preliminary study, we suggest that these fungal endophytes could prove beneficial for enhancing the salt stress tolerance of wheat crop.

Differential Symptom Development and Viral RNA Loads in 10 Nicotiana benthamiana Accessions Infected with the Tobamovirus Yellow Tailflower Mild Mottle Virus.

Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 (the lab type), which has a mutant, functionally defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done regarding responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harbored wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days postinoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried more than twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, in which viral RNA load was low until 15 dpi, after which it increased more than 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the 10 N. benthamiana accessions in some cases reflected geographical location, but in other accessions this was not so.

Beneficial effects of Trichoderma secondary metabolites on crops.

Selected microbial strains used as active ingredients of biopesticides for agricultural management practices (e.g., IPM, Integrated Pest Management) are known for their ability to control phytopathogens, promote plant growth, and/or induce disease resistance. Microbes belonging to the Trichoderma genus are considered as an appropriate example of beneficial microbes and are model organisms to study plant-microbe interactions. Several Trichoderma strains are marketed as biocontrol agents and are known to increase plant growth, stress tolerance, and nutrient availability. These effects have sometimes been related to the production of effector metabolites that beneficial microbes produce during the interaction with plant and other microbes. Secondary metabolites (SMs) comprise different classes of natural compounds with low molecular weight and having numerous biological roles, especially in the interactions among organisms. Metabolomic analysis of the interactions between plants, phytopathogens, and beneficial fungi aided in the identification of several bioactive fungal SMs that positively affect plant metabolism. Some of these compounds showed direct activity against phytopathogens, but also increased disease resistance by triggering the plant defence system, and/or enhanced vegetative growth. A new generation of bioformulations based on microbial metabolites and living consortia responsible for the desired beneficial effects on crops may overcome the difficulties associated with the use of a single living microbial strain.

Host Specificity of Endophytic Mycobiota of Wild Nicotiana Plants from Arid Regions of Northern Australia.

In arid regions of northern Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morpho-physiological and biochemical adaptations including interaction with microbial symbionts. We evaluated identity, host and tissue association with geographical distribution of fungal endophytes isolated from above- and below-ground tissues of plants of three indigenous Australian Nicotiana species. Isolation frequency and α-diversity were significantly higher for root endophyte assemblages than those of stem and leaf tissues. We recorded no differences in endophyte species richness or diversity as a function of sampling location, but did detect differences among different host genotypes and plant tissues. There was a significant pattern of community similarity associated with host genotypes but no consistent pattern of fungal community structuring associated with sampling location and tissue type, regardless of the community similarity measurements used.

A simple and rapid in vitro test for large-scale screening of fungal endophytes from drought-adapted Australian wild plants for conferring water deprivation tolerance and growth promotion in Nicotiana benthamiana seedlings.

Some fungal endophytes confer novel phenotypes and enhance existing ones in plants, including tolerance to water deprivation stress. A range of fungal endophytes was isolated from wild Nicotiana plants growing in arid parts of northern Australia. These were screened for ability to enhance water deprivation stress tolerance by inoculating seedlings of the model plant N. benthamiana in two in vitro tests. Sixty-eight endophyte isolates were co-cultivated with N. benthamiana seedlings on either damp filter paper or on agar medium before being subjected to water deprivation. Seventeen isolates were selected for further testing under water deprivation conditions in a sand-based test in a glasshouse. Only two fungal isolates, Cladosporium cladosporioides (E-162) and an unknown fungus (E-284), significantly enhanced seedling tolerance to moisture deprivation consistently in both in vitro and sand-based tests. Although a strongly significant correlation was observed between any two screening methods, the result of filter paper test was more strongly reflected (r = 0.757, p < 0.001) in results of the glasshouse test, indicating its relative suitability over the agar-based test. In another experiment, the same 17 isolates carried forward to the sand-based test used in the glasshouse screening test were inoculated to N. benthamiana plants in pots in a nutrient-limiting environment to test their influence on growth promotion. Isolates related to C. cladosporioides, Fusarium equiseti, and Thozetella sp. promoted seedling growth by increasing shoot length and biomass. The fungal isolate E-162 (C. cladosporioides) significantly enhanced moisture deprivation tolerance as well as promoted seedling growth.

A novel member of the Tombusviridae from a wild legume, Gompholobium preissii.

As part of an investigation into viruses of wild plants in Australia, a contiguous sequence of 3935 nucleotides was obtained after shotgun sequencing of RNA isolated from an asymptomatic wild legume, Gompholobium preissii. Phylogenetic analysis of the sequence revealed that it most closely resembled that of Trailing lespedeza virus 1 (TLV1), a virus isolated from a wild legume in America. The proposed virus, named Gompholobium virus A, and TLV1 are genetically closest to viruses in the genera Alphacarmovirus and Pelarspovirus, family Tombusviridae, but they share features distinguishing them from both groups.

Complete genome analysis of three isolates of narcissus late season yellows virus and two of narcissus yellow stripe virus: three species or one?

Complete genome sequences of two new isolates of narcissus late season yellows virus (NLSYV) from Australia were compared with the other NLSYV genome from China and with two complete genomes of isolates designated narcissus yellow stripe virus (NYSV), one from Australia and the other from China. On the basis of symptoms on natural and experimental host species, and genome sequence identity, the isolates could either be classified as closely related members of three different species or placed together in one taxon. Options for classification of these potyvirus isolates are discussed.

Spillover of a Tobamovirus from the Australian Indigenous Flora to Invasive Weeds.

The tobamovirus yellow tailflower mild mottle virus (YTMMV) was previously reported in wild plants of Anthocercis species (family Solanaceae) and other solanaceous indigenous species growing in natural habitats in Western Australia. Here, we undertook a survey of two introduced solanaceous weeds, namely Solanum nigrum (black nightshade) and Physalis peruviana (cape gooseberry) in the Perth metropolitan area and surrounds to determine if YTMMV has spread naturally to these species. At a remnant natural bushland site where both solanaceous weeds and indigenous Anthocercis hosts grew adjacent to one another, a proportion of S. nigrum and P. peruviana plants were asymptomatically-infected with YTMMV, confirming spillover had occurred. Populations of S. nigrum also grow as weeds in parts of the city isolated from remnant bushland and indigenous sources of YTMMV, and some of these populations were also infected with YTMMV. Fruit was harvested from virus-infected wild S. nigrum plants and the seed germinated under controlled conditions. Up to 80% of resultant seedlings derived from infected parent plants were infected with YTMMV, confirming that the virus is vertically-transmitted in S. nigrum, and therefore infection appears to be self-sustaining in this species. This is the first report of spillover of YTMMV to exotic weeds, and of vertical transmission of this tobamovirus. We discuss the roles of vertical and horizontal transmission in this spillover event, and its implications for biosecurity.

Proteome analysis of the Albugo candida-Brassica juncea pathosystem reveals that the timing of the expression of defence-related genes is a crucial determinant of pathogenesis.

White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell's immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida-Brassica juncea pathosystem, especially in relation to host resistance to this pathogen.

Host Range and Phylogenetic Relationships of Albugo candida from Cruciferous Hosts in Western Australia, with Special Reference to Brassica juncea.

White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) worldwide and poses a potential hazard to the presently developing canola-quality B. juncea industry in Australia. Nine isolates of A. candida, representing strains collected from B. juncea, B. rapa, B. oleracea, B. tournefortii, Raphanus raphanistrum, R. sativa, Eruca vesicaria subsp. sativa, Capsella bursa-pastoris and Sisymbrium irio, from different locations in Western Australia (W.A.), were tested on cruciferous host differentials to characterize their pathogenicity. In particular, these studies were aimed to determine the hazard to the newly emerging B. juncea industry in Australia from races or pathotypes of A. candida present. Pathogenicity tests with appropriate differentials demonstrated the presence in W.A. of a unique strain from B. rapa that did not show characteristics of either race 7A or 7V and clearly is a distinct new pathogenic strain within race 7. Different strains collected from W.A. differed in their host range, with the strains from B. tournefortii and S. irio being highly host specific, failing to be pathogenic on any other differentials. B. tournefortii was host to a strain attacking B. juncea and E. vesicaria subsp. sativa. The strain from R. raphanistrum showed a relatively wide host range among the differentials tested. B. tournefortii, C. bursa-pastoris, R. raphanistrum, and S. irio are common weeds within grain belt and horticultural regions in Australia. The B. oleracea isolate (race 9) was pathogenic to B. juncea 'Vulcan' whereas the isolate from B. juncea (race 2V) was not pathogenic on B. oleracea. Similarly, the strain from C. bursa pastoris (race 4) was pathogenic on B. juncea Vulcan but the B. juncea strain was not pathogenic on C. bursa pastoris. In contrast, the strain from R. sativus (race 1) was pathogenic on B. juncea and the B. juncea strain was also pathogenic on R. sativus. Field isolates from B. rapa, B. tournefortii, E. vesicaria subsp. sativa, and S. irio were all nonpathogenic on B. juncea. Isolates from B. juncea and R. raphanistrum were pathogenic on B. napus (FAN 189). For the nine A. candida isolates from W.A., complete rDNA internal transcribed spacer region nucleotide sequence analysis showed a nucleotide identity range of 72.4 to 100% in comparison with previous Australian collections of A. candida and those previously reported in Europe and Asia. The B. tournefortii isolate of A. candida from W.A. formed a distinct clade on its own, with an identity range of 77.4 to 80.5% compared with the other isolates. Isolates from R. raphanistrum and R. sativus from W.A. were least similar to the other isolates, with a nucleotide identity similarity of only 72.4%. Characterization of the races of A. candida in Western Australia adds to the current knowledge regarding the diversity of this pathogen, allows choice of Brassica spp. or cultivars with resistance to races across different regions, and highlights the particular cruciferous weeds involved in pathogen inoculum carryover between successive cruciferous crops, particularly B. juncea crops.

Metabolites of a Drechslera sp. endophyte with potential as biocontrol and bioremediation agent.

Endophytic fungi have several well-established beneficial effects on plant health and growth, and are a huge source of bioactive compounds. The endophyte Drechslera sp. strain 678, isolated from the roots of an Australian native grass Neurachne alopecuroidea, demonstrated efficacy against four plant pathogens (Pythium ultimum, Rhizoctonia solani, Botrytis cinerea, Alternaria alternata). In addition, strain 678 was capable of degrading a common additive used in gasoline, known as methyl tertiary-butyl ether (MtBE). Thus, the organic extracts obtained from the culture filtrate of strain 678 were studied. Metabolomic analysis revealed the presence of two major bioactive metabolites, monocerin and an alkynyl substituted epoxycyclohexenone derivative, which showed good antifungal activity. The Drechslera sp. strain 678 and its compounds show promise for applications in biocontrol and bioremediation activities in agriculture or as a remediation option for MtBE contamination in soil.

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus.

BACKGROUND AND AIMS: Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host-pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus). METHODS: Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar 'Charlton' and one susceptible 'RQ001-02M2' by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi). KEY RESULTS: The resistant 'Charlton' impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in 'Charlton' pathogen invasion was mainly confined to the upper epidermis, in the susceptible 'RQ001-02M2', colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible 'RQ001-02M2' at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant 'Charlton', crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in 'RQ001-02M2'. CONCLUSIONS: This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.

Comparison of the coat protein genes of Lettuce big-vein associated virus isolates from Australia with those of isolates from other continents.

The complete coat protein (CP) nucleotide sequences of seven Lettuce big-vein associated virus (LBVaV) isolates from Australia were compared to those of 22 other LBVaV and five tobacco stunt virus (TStV) isolates. On phylogenetic analysis, clade I contained only LBVaV isolates from Europe, sub-clade IIa only Australian LBVaV isolates, IIb only Japanese LBVaV isolates, and IIc only TStV isolates from Japan. In the amino acid sequences deduced, the central region of the gene was most divergent. Mean Dn/Ds ratios were 0.283 and 0.124 for clades I and II, respectively. The suggestion that TStV is a strain of LBVaV was supported.

Comparison of the coat protein genes of Mirafiori lettuce big-vein virus isolates from Australia with those of isolates from other continents.

The complete coat protein nucleotide encoding sequences of 13 Mirafiori lettuce big-vein virus isolates from Australia were compared to those of 23 other isolates, including one from Australia. On phylogenetic analysis, sub-clade A1 contained isolates from Australia (13), Europe and Japan, A2 contained isolates from Australia (1), Europe and South America, and B1 and B2 contained only European isolates. In the amino acid sequences deduced, the N-terminus and central regions varied considerably between clades A and B. Mean Dn/Ds ratios were 0.112, 0.076, 0.187 and 0.063 for all isolates, Australian isolates, clade A isolates and clade B isolates, respectively.

Scarification and Environmental Factors that Enhance Carpogenic Germination of Sclerotia of Sclerotinia sclerotiorum.

Ascospores of Sclerotinia sclerotiorum are the primary source of inoculum for disease epidemics in many economically important crops. Mass production of ascospores under laboratory conditions is required to prepare inoculum for use in selection of genotypes with resistance against Sclerotinia diseases. A study was undertaken, first, to investigate the effect on carpogenic germination of scarifying sclerotia from two S. sclerotiorum isolates taken from canola (Brassica napus) and, second, to identify environmental factors that enhance carpogenic germination. Seven different environmental treatments were applied to scarified and unscarified sclerotia: (i) sterilized distilled water for 4 months at 15°C, (ii) aerated water for 4 months at 4°C, (iii) constant rinsing with tap water for 8 weeks at 4°C, (iv) cold-conditioning for 4 weeks at 4°C and subsequent transfer into moist unsterilized compost at 15°C, (v) incubation in sterilized river sand at 15°C, (vi) air drying for 2 weeks followed by subsequent transfer into sterilized moist river sand at 15°C, or (vii) placed into 0.5% water agar and incubated at 15°C. Carpogenic germination of scarified sclerotia was significantly greater (P < 0.05) than for unscarified sclerotia. There was significant interaction (P < 0.001) between scarification and the different environmental treatments in relation to the carpogenic germination. Carpogenic germination of scarified sclerotia was enhanced by incubation of sclerotia in compost or in sterilized river sand. Further, overall carpogenic germination of both scarified and unscarified sclerotia occurred to the greatest extent when sclerotia of either of the two isolates were subjected to constant rinsing with tap water. We believe this to be the first report of both the enhanced carpogenic germination by scarification in S. sclerotiorum and the environmental factors we report that enhance carpogenic germination of scarified sclerotia. The progression of carpogenic germination in all the environmental treatments was also monitored as a part of this study across the two consecutive years for the same two isolates. The majority of sclerotia of both isolates germinated between the months of June and September in both years, a period which coincides with the main part of the cropping season when Sclerotinia stem rot is normally observed in rainfed canola in Western Australia. These data suggested the existence of a seasonal rhythm-like pattern in relation to the carpogenic germination of this pathogen.

Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum.

A Trichoderma harzianum strain, isolated from composted hardwood bark in Western Australia, was found to produce a metabolite with antifungal and plant growth promoting activity. The structure and absolute configuration of the fungal compound, harzianic acid (1), were determined by X-ray diffraction studies. Harzianic acid showed antibiotic activity against Pythium irregulare, Sclerotinia sclerotiorum, and Rhizoctonia solani. A plant growth promotion effect was observed at low concentrations of 1.

Novel and divergent viruses associated with Australian orchid-fungus symbioses.

Terrestrial orchids represent a symbiotic union between plants and mycorrhizal fungi. This study describes the occurrence and nature of viruses associated with one population of wild Pterostylis sanguinea orchids, including their fungal symbionts, over two consecutive years. A generic sequencing approach, which combined dsRNA-enrichment from plant and mycelial tissues, random amplification and high throughput shotgun sequencing was used to identify novel viruses. The majority of the virus-like sequences represent partial genomes, and their identification is based solely on de novo assembly of sequencing data. In orchid leaf tissues we found three isolates of a novel totivirus and an unclassified virus; both resemble fungus-infecting viruses. Two isolates of Ceratobasidium sp that were isolated from orchid underground stems contained at least 20 viruses, 16 of which were previously described as alphapartitiviruses and betapartitiviruses. A novel hypovirus and a mitovirus were genetically distant from existing members of the genera and did not readily fit into recognised subgroups.

The challenges of using high-throughput sequencing to track multiple bipartite mycoviruses of wild orchid-fungus partnerships over consecutive years.

The bipartite alpha- and betapartitiviruses are recorded from a wide range of fungi and plants. Using a combination of dsRNA-enrichment, high-throughput shotgun sequencing and informatics, we report the occurrence of multiple new partitiviruses associated with mycorrhizal Ceratobasidium fungi, themselves symbiotically associated with a small wild population of Pterostylis sanguinea orchids in Australia, over two consecutive years. Twenty-one partial or near-complete sequences representing 16 definitive alpha- and betapartitivirus species, and further possible species, were detected from two fungal isolates. The majority of partitiviruses occurred in fungal isolates from both years. Two of the partitiviruses represent phylogenetically divergent forms of Alphapartitivirus, suggesting that they may have evolved under long geographical isolation there. We address the challenge of pairing the two genomic segments of partitiviruses to identify species when multiple partitiviruses co-infect a single host.

Evolution of a wild-plant tobamovirus passaged through an exotic host: Fixation of mutations and increased replication.

Tobamovirus is a group of viruses that have become serious pathogens of crop plants. As part of a study informing risk of wild plant virus spill over to crops, we investigated the capacity of a solanaceous-infecting tobamovirus from an isolated indigenous flora to adapt to new exotic hosts. Yellow tailflower mild mottle virus (YTMMV) (genus Tobamovirus, family Virgaviridae) was isolated from a wild plant of yellow tailflower (Anthocercis littoria, family Solanaceae) and initially passaged through a plant of Nicotiana benthamiana, then one of Nicotiana glutinosa where a single local lesion was used to inoculate a N. benthamiana plant. Sap from this plant was used as starting material for nine serial passages through three plant species. The virus titre was recorded periodically, and 85% of the virus genome was sequenced at each passage for each host. Six polymorphic sites were found in the YTMMV genome across all hosts and passages. At five of these, the alternate alleles became fixed in the viral genome until the end of the experiment. Of these five alleles, one was a non-synonymous mutation (U1499C) that occurred only when the virus replicated in tomato. The mutant isolate harbouring U1499C, designated YTMMV-δ, increased its titre over passages in tomato and outcompeted the wild-type isolate when both were co-inoculated to tomato. That YTMMV-δ had greater reproductive fitness in an exotic host than did the wild type isolate suggests YTMMV evolution is influenced by host changes.

An antifungal and plant growth promoting metabolite from a sterile dark ectotrophic fungus.

A sterile dark ectotrophic fungus isolated from roots of an Australian native grass, Neurachne alopecuroidea produces compound 1 in liquid cultures. The structure of the metabolite was determined by spectroscopic and X-ray diffraction studies. The metabolite shows activity against phytopathogens and plant growth promoting activity, properties that are also expressed in vivo by the ectotrophic fungus.