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Ginger (Zingiber officinale Roscoe) is an important horticulture crop valued for its medicinal and culinary properties. Fusarium yellows, caused by the ascomycete fungus Fusarium oxysporum f. sp. zingiberi (Foz) is a devastating soil borne disease of ginger. It has curtailed ginger production in Australia and around the world leading to significant economic losses. An integrated approach is required to manage soil-borne diseases such as those caused by Foz. Little is known about the influence of Foz inoculum dosage on disease severity. This study aimed to establish a minimum threshold level of spores per gram of soil required for plant infection and to develop and evaluate a pot inoculation method for suitability in genetic studies on Foz resistance. To achieve this, the dominant Australian ginger cultivar ‘Canton’ was inoculated with pre-determined amounts of colony-forming unit (cfu), ranging from 0 in control, to treatments with 10, 103, 105, 106, and 107 cfu per gram of soil. The inoculum dosage positively correlated with leaf and stem yellows, and rhizome discolouration, and negatively correlated with root length and rhizome weight. The lowest threshold required for plant infection was 10 cfu per gram of soil, which may provide an important basis for outbreaks of Foz in the field. This finding adds significantly to the knowledge base on ginger soil health and contributes to the integrated management of Foz. When used at a high dose, this method can facilitate reliable and accurate screening of Foz susceptible ginger genotypes in a controlled environment.

Fusarium crown rot (FCR) caused by the fungus Fusarium pseudograminearum (Fp) is an important disease of wheat that reduces yield and grain quality in many countries, including Australia. In this study, we investigated mating type idiomorph composition, putative chemotype and population genotypic structure of 98 Fp isolates from Western Australia (WA) and the eastern Australian states of New South Wales, Victoria and South Australia. Our results revealed the expected 1:1 mating type ratio for isolates from the eastern states while there was significant variation to a 1:1 mating type composition with isolates from WA. A chemotype-specific PCR assay indicated that all Fp isolates from eastern states and WA segregated for the 3-ADON trichothecene chemotype. Genetic diversity assessed using 21 cleaved amplified polymorphic sequence markers revealed a high level of genotypic variation within and between Fp populations from eastern Australian states and WA. Analysis of molecular variance (AMOVA) showed significant difference between populations from eastern states and WA. The genetic diversity measured by Shannon index ranged from 0.95 to 2.30 with the lowest and highest values detected in Hart and Rowena populations, respectively in the eastern states. Index of association analysis showed no significant linkage of markers among isolates within 60% of the populations, suggesting that sexual reproduction may be occurring in the pathogen from those locations. These results improve understanding of Fp population dynamics across Australia and highlight the importance of monitoring for shifts in the population which could have implications for disease management.

The ecology of Tribolium castaneum (Herbst) outside of grain storage facilities is poorly known. However, high densities of T. castaneum adults are known to infest stored cotton seeds (Gossypium hirsutum (L.)) in Queensland, Australia, despite the absence of stored food products in the immediate vicinity. Previous studies suggest that the beetles are attracted to the fungal colonies growing on the residual fibres that remain on the cotton seeds after ginning, but the specifics of this remain unclear, even as to the species of fungi involved. In the current study, 14 fungal species were isolated from stored cotton seeds collected from four sites in Queensland. The feeding preferences of the adult beetles and the developmental success of the larvae were recorded for each fungal species. Gut analyses of T. castaneum adults, after exposure in no-choice feeding tests, showed that the beetles will feed on any one of the 14 fungal species when exposed (over 14 days) to cotton seeds that had been inoculated with one particular fungal species. The developmental success of the larvae varied depending on the fungal species, with most fungal species supporting only low levels of successful development (

Robust antifungal screening is technically challenging particularly for filamentous fungi. We present a method for undertaking antifungal screening assays that builds upon existing broth dilution protocols and incorporates time resolved image-based assessment of fungal growth. We show that the method performs with different fungi, particularly those for which spores can be used as inoculum, and with different compound classes, can accurately assess susceptibility or otherwise in only few hours, performs well even without replication and can even account for differences in inherent growth properties of strains.

Robust antifungal screening is technically challenging particularly for filamentous fungi. We present a method for undertaking antifungal screening assays that builds upon existing broth dilution protocols and incorporates time resolved image-based assessment of fungal growth. We show that the method performs with different fungi, particularly those for which spores can be used as inoculum, and with different compound classes, can accurately assess susceptibility or otherwise in only few hours and can even account for differences in inherent growth properties of strains.

Macrophomina phaseolina causes charcoal rot in strawberry. The pathogen has a wide host range, is favored by high soil temperatures, and current fumigants are not as effective as methyl bromide. Breeding strawberry cultivars resistant to M. phaseolina has become an important focus. Eleven cultivars were evaluated in a glasshouse trial for resistance to an isolate of M. phaseolina. Plants were inoculated by drenching the potting medium with a suspension of microsclerotia. Plant mortality was recorded for up to 23 weeks. Based on plant mortality and survival analyzes, ‘Albion’ was similarly susceptible as ‘Camarosa’ and a number of historical and current cultivars showed tolerance and/or resistance to the pathogen. The preliminary findings in this study can assist in the development of new strawberry genotypes against M. phaseolina.

Despite being recognised as a serious pest of turf grasses in Australia, there has been limited research into the southern sting nematode (Ibipora lolii). This study examined its occurrence and population dynamics in the Perth region of Western Australia and assessed the level of resistance and tolerance of turf grasses to this nematode. In a systematic survey of 90 municipal sports fields on which kikuyu was the most common turf species, I. lolii was detected in 51% of sites. The mean population density was 27 nematodes/200 mL soil, but was almost eight times higher in localised areas where symptoms were severe. Infested turf tended to be relatively sparse and have a shallow root system. In a 2-year population dynamics study on a kikuyu sports field, I. lolii reproduced year-round, but exhibited a seasonal pattern of fluctuation in population density. Numbers increased when conditions were cool and wet during the winter rainfall period, peaking in October, and then declined during the hot and dry irrigation season. Although the population density of I. lolii was always greatest in the upper 10 cm of the soil profile, nematodes were consistently recovered from depths of 50–70 cm, showing that it was capable of deep vertical movement. A field experiment comparing four cultivars of couch and two cultivars of kikuyu showed that none of the cultivars exhibited resistance or tolerance to I. lolii. Nematode numbers increased rapidly in infested plots, regardless of cultivar, while in comparison with non-infested plots, the mean total plant biomass and root dry weight were reduced by 53% and 88%, respectively.

During the infection of a host, plant pathogenic fungi secrete small proteins called effectors, which then modulate the defence response of the host. In the Fusarium oxysporum species complex (FOSC), the secreted in xylem (SIX) gene effectors are important for host-specific pathogenicity, and are also useful markers for identifying the various host-specific lineages. While the presence and diversity of the SIX genes has been explored in many of the pathogenic lineages of F. oxysporum, there is a limited understanding of these genes in non-pathogenic, endophytic isolates of F. oxysporum. In this study, universal primers for each of the known SIX genes are designed and used to screen a panel of endophytically-associated Fusarium species isolated from healthy, asymptomatic banana tissue. SIX gene orthologues are identified in the majority of the Fusarium isolates screened in this study. Furthermore, the SIX gene profiles of these endophytic isolates do not overlap with the SIX genes present in the pathogenic lineages of F. oxysporum that are assessed in this study. SIX gene orthologues have not been commonly identified in Fusarium species outside of the FOSC nor in non-pathogenic isolates of F. oxysporum. The results of this study indicate that the SIX gene effectors may be more broadly distributed throughout the Fusarium genus than previously thought. This has important implications for understanding the evolution of pathogenicity in the FOSC.

Members of the Fusarium oxysporum species complex include pathogenic and non-pathogenic isolates and infect a broad range of plant species. F. oxysporum f. sp. cubense (Foc) causes the destructive Fusarium wilt of banana, and the recently emerged Foc tropical race 4 strain threatens the global banana industry. Secreted in xylem (SIX) genes encode for F. oxysporum effector proteins that are associated with virulence in pathogenic F. oxysporum, however they have rarely been reported from non-pathogenic F. oxysporum isolates. Our recent survey of asymptomatic banana plants grown in Foc-infested fields in Queensland and northern NSW revealed that diverse Fusarium spp, including F. oxysporum, reside in the plant roots and pseudostem without causing obvious damage to the plant. Intriguingly, we amplified SIX genes from several of the putative endophytic F. oxysporum isolates identified in the survey and found that they differ in their profile to known Foc SIX genes. To study the role of the endophytic F. oxysporum isolates in planta and the biological function of their SIX genes in more detail, we will re-inoculate cultivated and wild diploid banana lines with the endophytic F. oxysporum strains under glasshouse conditions to assess if they are non-pathogenic on banana. Secondly, we will determine whether the endophytic F. oxysporum SIX genes are expressed in planta and/or in vitro and look at the transcriptome changes occurring in the host following infection. Finally, endophytic F. oxysporum strains transformed with GFP will be used to investigate the extent of fungal colonisation in the plant.

In 2011, patches of lodged plants of Helianthus annuus (commercial sunflower) with stem lesions were observed in a commercial crop at Kingsthorpe, Queensland, Australia. Several species of Diaporthe were consistently isolated from the lesions. Diaporthe novem was identified by DNA sequence analysis. In following surveys, Diaporthe novem was found to be associated with other crop species including Cicer arietinum (chickpea), Glycine max (soybean), Lupinus alba (lupin), Sorghum bicolor (sorghum) Vicia faba (faba bean) and Vigna radiata (mungbean), and with the weed species Datura stramonium (common thornapple), Helianthus annuus (wild-type sunflower), Malva parviflora (small flowered mallow), Rapistrum rugosum (turnip weed), Sambucus gaudichaudiana (wild elderberry), Sisymbrium orientale (indian hedge mustard), Sonchus oleraceus (sowthistle), Verbena sp., Vicia sativa (common vetch), and Xanthium strumarium (noogoora burr). In pathogenicity tests, isolates of D. novem from sunflower were highly virulent when re-inoculated on commercial sunflower varieties. This study has identified D. novem as a frequent cause of stem canker of sunflower in the eastern cropping areas of Australia, and extends the known host range of D. novem.

Banana is an important tropical crop and trading commodity in the world. However, Fusarium wilt is a severe risk for the world banana industry. Caused by the soil-borne fungal pathogen, Fusarium oxysporum f. sp. cubense (Foc), the disease is spreading insidiously and has been a source of enormous losses worldwide since the 1800s. Unfortunately, effective control methods are not available yet. The current study used an RNAi strategy, referred to as host-induced gene silencing (HIGS), to confer durable resistance against Fusarium by inhibiting in planta expression of Foc essential genes that are conserved in all Foc races and crucial for fungal virulence. In order to find efficient target genes, a wide search of literature has been conducted and four conserved target regions have been chosen after bioinformatics analysis. Preliminary findings from in vitro fungal inhibition assays showed significant reductions in fungal spore count, germination rate and mycelia growth after treating fungal cultures with double-stranded RNA (dsRNA) targeting four Foc genes, suggesting silencing of these genes was effective to reduce fungal growth. Intron hairpin constructs encoding dsRNA targeting selected Foc genes are in the process of being transformed into Arabidopsis thaliana for proof-of-concept of in planta fungal resistance.

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana-infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole-genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1-SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.

Thirteen potentially new leaf rust resistance loci were identified in a Vavilov wheat diversity panel. We demonstrated the potential of allele stacking to strengthen resistance against this important pathogen. Leaf rust (LR) caused by Puccinia triticina is an important disease of wheat (Triticum aestivum L.), and the deployment of genetically resistant cultivars is the most viable strategy to minimise yield losses. In this study, we evaluated a diversity panel of 295 bread wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources (St Petersburg, Russia) for LR resistance and performed genome-wide association studies (GWAS) using 10,748 polymorphic DArT-seq markers. The diversity panel was evaluated at seedling and adult plant growth stages using three P. triticina pathotypes prevalent in Australia. GWAS was applied to 11 phenotypic data sets which identified a total of 52 significant marker–trait associations representing 31 quantitative trait loci (QTL). Among them, 29 QTL were associated with adult plant resistance (APR). Of the 31 QTL, 13 were considered potentially new loci, whereas 4 co-located with previously catalogued Lr genes and 14 aligned to regions reported in other GWAS and genomic prediction studies. One seedling LR resistance QTL located on chromosome 3A showed pronounced levels of linkage disequilibrium among markers (r 2 = 0.7), suggested a high allelic fixation. Subsequent haplotype analysis for this region found seven haplotype variants, of which two were strongly associated with LR resistance at seedling stage. Similarly, analysis of an APR QTL on chromosome 7B revealed 22 variants, of which 4 were associated with resistance at the adult plant stage. Furthermore, most of the tested lines in the diversity panel carried 10 or more combined resistance-associated marker alleles, highlighting the potential of allele stacking for long-lasting resistance.

Pythium myriotylum is responsible for severe losses in both capsicum and ginger crops in Australia under different regimes. Intraspecific genomic variation within the pathogen might explain the differences in aggressiveness and pathogenicity on diverse hosts. In this study, whole genome data of four P. myriotylum isolates recovered from three hosts and one Pythium zingiberis isolate were derived and analysed for sequence diversity based on single nucleotide polymorphisms (SNPs). A higher number of true and unique SNPs occurred in P. myriotylum isolates obtained from ginger with symptoms of Pythium soft rot (PSR) in Australia compared to other P. myriotylum isolates. Overall, SNPs were discovered more in the mitochondrial genome than those in the nuclear genome. Among the SNPs, a single substitution from the cytosine (C) to the thymine (T) in the partially sequenced CoxII gene of 14 representatives of PSR P. myriotylum isolates was within a restriction site of HinP1I enzyme which was used in the PCR-RFLP for detection and identification of the isolates without sequencing. The PCR-RFLP was also sensitive to detect PSR P. myriotylum strains from artificially infected ginger without the need for isolation for pure cultures.This is the first study of intraspecific variants of Pythium myriotylum isolates recovered from different hosts and origins based on single nucleotide polymorphism (SNP) genotyping of multiple genes. The SNPs discovered provide valuable makers for detection and identification of P. myriotylum strains initially isolated from Pythium soft rot (PSR) ginger by using PCR-RFLP of the CoxII locus. The PCR-RFLP was also sensitive to detect P. myriotylum directly from PSR ginger sampled from pot trials without the need of isolation for pure cultures.

Historical records report Fusarium moniliforme sensu lato as the pathogen responsible for Fusarium diseases of sorghum; however, recent phylogenetic analysis has separated this complex into more than 25 species. During this study, surveys were undertaken in three major sorghum-producing regions in eastern Australia to assess the diversity and frequency of Fusarium species associated with stalk rot- and head blight-infected plants. A total of 523 isolates were collected from northern New South Wales, southern Queensland and central Queensland. Nine Fusarium species were isolated from diseased plants. Pathogenicity tests confirmed F. andiyazi and F. thapsinum were the dominant stalk rot pathogens, whilst F. thapsinum and species within the F. incarnatum–F. equiseti species complex were most frequently associated with head blight.

Pythium myriotylum and P. zingiberis have both been implicated in soft rot of ginger (Zingiber spp.). The status of these two taxa as distinct species follows original descriptions of physiology, morphology and pathogenicity. However, their status has been questioned by phylogenetic analyses. In this study, putative P. zingiberis isolates recovered from edible ginger (Z. officinale) rhizomes with Pythium soft rot (PSR) disease sampled from Queensland, Australia, were compared with reference isolates of P. myriotylum from peanut in Israel (CBS254.70) and P. zingiberis from ginger in Japan (NBRC30817). All isolates differed slightly in temperature optima for growth and produced similar sizes of oogonia and oospores. The sequence homology of 20 gene fragments retrieved from nuclear and mitochondrial genomes ranges from over 99 to 100% to each other. In vitro pathogenicity assays were conducted on excised carrot, ginger, potato, radish and sweet potato tuber/root sections and on seeds and seedlings of cucumber, cauliflower, millet, rye, sweet corn, tomato and wheat with each of the isolates. The reference isolate P. zingiberis NBRC30817, which was previously believed to have a narrow host range, was pathogenic on a number of tested plant species. Analysis of this comprehensive set of data allowed us to assign all tested isolates, including the isolate P. zingiberis NBRC30817, to the taxon P. myriotylum, thus confirming that the causal pathogen of PSR disease of ginger in Australia is P. myriotylum.

Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.

Fusarium oxysporum f. sp. cubense (Foc) has severely curtailed banana production in the tropical regions of the world. The tropical race 4 (TR4) of Foc was detected in Australia in the 1990s and it is virulent to all Cavendish type banana cultivars, which represents the majority of banana production in Australia. Genetic resistance to Foc race 4 is urgently needed. To characterize sources of resistance, we have assessed the Foc resistance response of 34 Musa cultivars with plants grown under controlled settings. Amongst diploid banana cultivars carrying the AA genome, resistance is found in Musa acuminata sub-species including malaccensis ‘Pahang’ and burmannica ‘Calcutta4.’ In the polyploid group, the hybrids such as ‘FHIA-18’ and ‘FHIA-25’ are highly resistant against both Foc-TR4 and subtropical race 4 (Foc-STR4). Interestingly, ‘FHIA-2’ and ‘CAM020’ appear to be resistant to Foc-TR4 but susceptible to Foc-STR4, suggesting potential differences in the resistance mechanisms against the different race 4 strains. Using a GFP tagged Foc-STR4 strain challenged onto both resistant and susceptible M. a. malaccensis lines, a high inoculum dosage rapidly induced vascular wilt in the susceptible M. a. malaccensis lines at 2.5 weeks. This was associated with an accumulation of micro-conidia in the rhizome and the movement of the fungus through the xylem vessels. In contrast, the fungal movement was restrained in the rhizome of the resistant M. a. malaccensis lines and no sporulation was observed. Overall, this research suggests that the resistance response is dependent to an extent on inoculum dosage and that the plant host’s response, in the rhizome, plays an important role in inhibiting the fungus from spreading to the rest of the plant. Identifying race 4 resistant accessions can help to understand mechanisms of resistance and provide banana breeders with the genetic resources to integrate resistance genes into commercial varieties.

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Muskmelon (Cucumis melo L.) is one of the most important vegetable crops in Oman. In the fall of 2004, sudden wilt was observed in muskmelon grown in a field at Sultan Qaboos University, Muscat. The disease was characterized by rapid collapse of vines and muskmelon plants at the fruit production to maturation stage, associated with brown-to-dark brown rotted primary and secondary roots. The disease resulted in death of more than 85% of muskmelon plants in that field. On potato dextrose agar (PDA), with published methods (1), Pythium spp. were consistently isolated from crowns and roots of plants showing wilt symptoms. Further identification of five isolates of Pythium with sequences of the internal transcribed spacer (ITS) of the ribosomal DNA (1) using ITS1 and ITS4 primers produced a nucleotide sequence 806 bp long, which was identical among all isolates. Comparison with sequences deposited at the National Center for Biotechnology Information revealed 100% nucleotide similarity to a previously published sequence (Accession No. DQ381808) of isolate P091 of P. splendens from cucumber from Oman, for which identification has also been confirmed by morphological characteristics. The sequence of one isolate of P. splendens (P222) was assigned GenBank Accession No. EF546436 and deposited at CBS under Accession No. CBS121855. In pathogenicity tests conducted in a greenhouse, P. splendens induced damping-off symptoms on 7-day-old muskmelon seedlings and also reproduced the same wilt symptoms observed in the field when 2-month-old muskmelon plants were inoculated with 3-day-old P. splendens grown in PDA. To our knowledge, this is the first report of association of P. splendens with wilt of muskmelon in Oman. Reference: (1) A. M. Al-Sa'di et al. Plant Pathol. 56:140, 2007.

The Old World climbing fern, Lygodium microphyllum (Cav.) R. Br., and Japanese climbing fern, L. japonicum (Thunb.) Sw., are invasive noxious weeds in Florida (1). Exploratory surveys for classical biological control agents of L. microphyllum in the fern's native range of Australia and Asia have focused on aboveground herbivores (1). From February to August 2006, fungi were isolated from symptomatic foliage, including lesions associated with leaf curls caused by the mite Flocarus perrepae Knihinicki & Boczek., obtained from L. microphyllum at sites across southeast Queensland, Australia and from both fern species grown at the CSIRO Long Pocket Laboratories in Brisbane, Australia. Anthracnose symptoms with chlorotic margins, initiating at the tip or base of the individual pinnules, were observed on fronds. Dieback symptoms affected growing tips, with sunken lesions and a gradual necrotic wilt as far as the next growth junction of pinnae. Sections from diseased margins were surface sterilized, placed onto water agar, and incubated at 23°C with a 16-h photoperiod. Variable colonies of white-to-gray mycelia, felted or tufted with complete margins, grew well on oatmeal agar and potato dextrose agar. Conidia were hyaline to light salmon, aseptate, straight, and cylindrical (10.4 to 18.2 × 2.6 to 5.2 μm), borne in salmon-to-bright orange masses at 25°C, and consistent with previous descriptions of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (3), anamorph of Glomerella cingulata (2). Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical (46.8 to 62.4 × 9.1 to 11.7 μm), and thickened at the apex, and ascospores were cylindrical (11.7 to 18.2 × 3.9 to 5.2 μm), slightly curved, unicellular and hyaline, which is consistent with descriptions of G. cingulata (2). No fruiting bodies were observed in planta; acervuli, setae, and perethecia were not observed. Identification was further confirmed by molecular analysis using the primer pair ITS1/ITS4 (4) (GenBank Accession No. EU697014), indicating 100% similarity to isolates of G. cingulata. To confirm pathogenicity, Koch's postulates were performed on three plants of L. japonicum and 12 plants of L. microphyllum, with an equal number of controls. Conidial suspensions were made to 1.7 × 106 conidia ml–1. During the experiments in the glasshouse, temperatures ranged from 12.6 to 40°C and relative humidity from 39 to 85%. Tips and fronds were collected after 2 to 8 weeks and isolation and identification performed. G. cingulata was consistently reisolated from diseased tissue. No symptoms appeared on controls and isolations did not yield the pathogen. To our knowledge, this is the first report of G. cingulata infecting L. microphyllum and L. japonicum in Australia. Its potential as a biological control agent in the ferns' introduced range remains to be tested. References: (1) J. A. Goolsby et al. Biol. Control. 28:33, 2003. (2) J. E. M. Mordue. Glomerella cingulata. No. 315 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CAB, Kew, UK, 1971. (3) B. C. Sutton. The Genus Glomerella and its Anamorph Colletotrichum. In: Colletotrichum: Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, UK, 1992. (4) T. M. White et al. Amplification and Direct Sequencing of Fungal Ribosomal RNA for Phylogenetics. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Fusarium wilt caused by the soil-borne fungus Fusarium oxysporum f.sp. cubense (Foc) is a significant constraint to banana production worldwide, with the recent expansion of banana growing regions impacted by Foc Tropical Race 4 (TR4). The lack of commercially acceptable Cavendish cultivars with Foc resistance means the only current means of effective control is through strict quarantine and inoculum management. One method of control that is currently advocated includes the removal of infected plants which have been killed using herbicide injections. The aim of this work was to examine the effect of herbicide and fungicide treatments on sporulation of the fungus. In glasshouse studies using a green fluorescent transformed Foc Subtropical Race 4 isolate, we found treatments with herbicide hastened colonisation of the banana tissue and the production of micro- and macroconidia. The use of a fungicide did not prevent sporulation of the fungus in such tissue. This study demonstrates that herbicide treated plants are a source of potential inoculum for infection of nearby plants.

Variation in the virulence of Fusarium oxysporum f. sp. fragariae (Fof) strains is important when evaluating the resistance of plants to this fungus. Twenty-five isolates of F. oxysporum harvested from strawberry (Fragaria ×ananassa) plants growing in Australia were characterized using pathogenicity tests, vegetative compatibility groups (VCGs), and genetic analysis of translation elongation factor 1 alpha (EF-1α). The level of disease varied depending on isolate used, indicating heterogeneous populations of Fof. Two distinct VCGs were identified and corresponded to two of the 10 lineages identified by partial EF-1α. Using a subset of Fof isolates, resistance in eight cultivars ranged from highly resistant to highly susceptible, with some cultivar × isolate interaction. ‘Strawberry Festival’, ‘QHI Sugarbaby’, and ‘DPI Rubygem’ had high levels of resistance across all isolates. Isolates from Western Australia (WA) were genetically distinct from those from Queensland (QLD) and were more virulent to ‘Camarosa’, a major cultivar grown in WA.

BACKGROUND AND AIMS: Silicon has been shown to enhance the resistance of plants to fungal and bacterial pathogens. Here, the effect of potassium silicate was assessed on two cotton (Gossypium hirsutum) cultivars subsequently inoculated with Fusarium oxysporum f. sp. vasinfectum (Fov). Sicot 189 is moderately resistant whilst Sicot F-1 is the second most resistant commercial cultivar presently available in Australia. METHODS: Transmission and light microscopy were used to compare cellular modifications in root cells after these different treatments. The accumulation of phenolic compounds and lignin was measured. KEY RESULTS: Cellular alterations including the deposition of electron-dense material, degradation of fungal hyphae and occlusion of endodermal cells were more rapidly induced and more intense in endodermal and vascular regions of Sicot F-1 plants supplied with potassium silicate followed by inoculation with Fov than in similarly treated Sicot 189 plants or in silicate-treated plants of either cultivar not inoculated with Fov. Significantly more phenolic compounds were present at 7 d post-infection (dpi) in root extracts of Sicot F-1 plants treated with potassium silicate followed by inoculation with Fov compared with plants from all other treatments. The lignin concentration at 3 dpi in root material from Sicot F-1 treated with potassium silicate and inoculated with Fov was significantly higher than that from water-treated and inoculated plants. CONCLUSIONS: This study demonstrates that silicon treatment can affect cellular defence responses in cotton roots subsequently inoculated with Fov, particularly in Sicot F-1, a cultivar with greater inherent resistance to this pathogen. This suggests that silicon may interact with or initiate defence pathways faster in this cultivar than in the less resistant cultivar.

Intensive selection in wheat (Triticum aestivum L.) breeding programs over the past 100 years has led to a genetic bottleneck in modern bread wheat. Novel allelic variation is needed to break the yield plateau, particularly in the face of climate change and rapidly evolving pests and pathogens. Landraces preserved in seed banks likely harbour valuable sources of untapped genetic diversity because they were cultivated for thousands of years under diverse eco-geographical conditions prior to modern breeding. We performed the first genetic characterisation of bread wheat accessions sourced from the N. I. Vavilov Institute of Plant Genetic Resources (VIR) in St Petersburg, Russia. A panel comprising 295 accessions, including landraces, breeding lines and cultivars was subject to single seed descent (SSD) and genotyped using the genotyping-by-sequencing Diversity Arrays Technology platform (DArT-seq); returning a total of 34,311 polymorphic markers (14,228 mapped and 20,083 unmapped). Cluster analysis identified two distinct groups; one comprising mostly breeding lines and cultivars, and the other comprising landraces. Diversity was benchmarked in comparison to a set of standards, which revealed a high degree of genetic similarity among breeding material from Australia and the International Maize and Wheat Improvement Center (CIMMYT). Further, 11,025 markers (1888 mapped and 9137 unmapped) were polymorphic in the diversity panel only, thus representing allelic diversity potentially not present in Australian or CIMMYT germplasm. Open-access to DArT-seq markers and seed for SSD lines will empower researchers, pre-breeders and breeders to rediscover genetic diversity in the VIR collection and accelerate utilisation of novel alleles to improve wheat.

Fusarium oxysporum f. sp. cubense (Foc) is a fungal pathogen of banana and is a significant threat to global banana production. A novel strain of Foc (named tropical race 4) first emerged in Southeast Asia and has recently been detected in Africa and Jordan. Tropical race 4 of Foc is of significant concern to the banana industry as it is extremely pathogenic towards all commercial banana cultivars. Currently, very few genes involved in pathogenicity have been identified in Foc. However, many pathogenicity-associated genes have been studied in the F oxysporum species complex. This study sought to identify the orthologues of genes previously demonstrated to be involved in the pathogenicity of F oxysporum in a collection of Foc isolates. Of those genes assessed, we detected all pathogenicity-associated genes in all Foc isolates, with the exception of SIX8, which was only detected in race 4 isolates. Our results indicate that many genes are required for general pathogenicity towards plants and are widely distributed across the F oxysporum species complex. However, the host-specific pathogenicity of Foc may be attributed to fungal effectors, such as SIX8. Previous work in the tomato-infecting lineage of E oxysporum has demonstrated that the SIX gene effector family is localized on a small, dispensable chromosome which has also been associated with the host-specific pathogenicity of the tomato infecting lineages. The current work sheds light on the pathogenomics of Fusarium. The results of this study are significant for the diagnosis and management of Foc. The identification and characterization of the genes controlling pathogenicity in Foc should be an ongoing field of research. It will also be of substantial value to better understand the genomic organization of each VCG of Foc for the development a pangenome of Foc and the E oxysporum species complex. An improved understanding of the pathogenomics of Foc is critical for the ongoing improvement of disease management strategies for Fusarium wilt of banana.

Fusarium wilt of banana, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. A particularly virulent strain of the pathogen, tropical race 4 (TR4), presents an emerging threat to banana producing regions throughout the world. No commercially acceptable banana cultivar is resistant to TR4 and, as with all strains of the Fusarium wilt pathogen, there is no effective chemical control. Genetic resistance to TR4 has been observed in the diploid wild banana Musa acuminata subsp. malaccensis, which has consequently received attention as a potential source of Fusarium resistance genes. The aim of this research was to determine the pattern of inheritance of the resistance trait by screening plants for resistance to Foc subtropical race 4 (SR4) and TR4. Our results showed that the F1 progeny of self-fertilized malaccensis plants challenged in pot trials against SR4 (VCGs 0120, 0129, 01211) and TR4 (VCG 01213/16) segregated for resistance according to a Mendelian ratio of 3:1 which is consistent with a single dominant gene hypothesis.

Leaf rust (LR) caused by Puccinia triticina, is among the most important diseases of wheat (Triticum aestivum L.) crops globally. Deployment of cultivars incorporating genetic resistance, such as adult plant resistance (APR) or all-stage resistance, is considered the most sustainable control method. APR is preferred for durability because it places lower selection pressure on the pathogen and is often polygenic. In the search for new sources of APR, here we explored a diversity panel sourced from the N. I. Vavilov Institute of Plant Genetic Resources. Based on DNA marker screening, 83 of the 300 lines were deemed to carry known APR genes; namely, Lr34, Lr46, and Lr67. Interestingly, lines carrying Lr67 were mostly landraces from India and Pakistan, reconfirming the likely origin of the gene. Rapid phenotypic screening using a method that integrates assessment at both seedling and adult growth stages under accelerated growth conditions (i.e., constant light and controlled temperature) identified 50 lines carrying APR. Levels of APR corresponded well with phenotypes obtained in a field nursery inoculated using the same pathotype (R2 = 0.82). The second year of field testing, using a mixture of pathotypes with additional virulence for race-specific APR genes (Lr13 and Lr37), identified a subset of 13 lines that consistently displayed high levels of APR across years and pathotypes. These lines provide useful sources of resistance for future research. A strategy combining rapid generation advance coupled with phenotyping under controlled conditions could accelerate introgression of these potentially novel alleles into adapted genetic backgrounds.

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Diaporthe (syn. Phomopsis) species are well-known saprobes, endophytes or pathogens on a range of plants. Several species have wide host ranges and multiple species may sometimes colonise the same host species. This study describes eight novel Diaporthe species isolated from live and/or dead tissue from the broad acre crops lupin, maize, mungbean, soybean and sunflower, and associated weed species in Queensland and New South Wales, as well as the environmental weed bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern Australia. The new taxa are differentiated on the basis of morphology and DNA sequence analyses based on the nuclear ribosomal internal transcribed spacer region, and part of the translation elongation factor-1α and s-tubulin genes. The possible agricultural significance of live weeds and crop residues ('green bridges') as well as dead weeds and crop residues ('brown bridges') in aiding survival of the newly described Diaporthe species is discussed.

Apart from morphology and genetic characteristics, species status of Pythium zingiberis and P. myriotylum may also be confirmed based on their pathogenicity and host range. An Australian putative P. zingiberis isolate and imported type isolates of P. myriotylum and P. zingiberis were subject to both in vitro and in vivo pathogenicity tests. In vitro tests were carried out on excised carrot, ginger, potato, radish, and sweet potato tuber/root sections, and on seeds and seedlings of cucumber, cauliflower, millet, rye, sweet corn, tomato, and wheat. In all assays conducted, the Australian isolate was found to be the most pathogenic, followed by type specimen of P. zingiberis (UOP 275), and then the type specimen P. myriotylum (CBS 254.70). An in vivo experiment on ginger plants at 35°C (with 10 h day light) in quarantine conditions showed that the ginger plants inoculated with the Australian isolate and also the type specimen of P. zingiberis died at 21 days after inoculation, whereas those inoculated with P. myriotylum CBS 254.70 were still green and healthy. Along with cardinal growth rate, the Australian isolate was confirmed to be closely related to P. zingiberis. This is also the first direct comparison in pathogenicity of P. zingiberis and P. myriotylum.

Pythium soft rot (PSR) of ginger caused by a number of Pythium species is of the most concern worldwide. In Australia, PSR outbreaks associated with Pythium myriotylum was recorded in 2007. Our recent pathogenicity tests in Petri dishes conducted on ginger rhizomes and pot trials on ginger plants showed that Pythiogeton (Py.) ramosum, an uncommon studied oomycete in Pythiaceae, was also pathogenic to ginger at high temperature (30–35 °C). Ginger sticks excised from the rhizomes were colonised by Py. ramosum which caused soft rot and browning lesions. Ginger plants inoculated with Py. ramosum showed initial symptoms of wilting and leave yellowing, which were indistinguishable from those of Pythium soft rot of ginger, at 10 days after inoculation. In addition, morphological and phylogenetic studies indicated that isolates of Py. ramosum were quite variable and our isolates obtained from soft rot ginger were divided into two groups based on these variations. This is also for the first time Py. ramosum is reported as a pathogen on ginger at high temperatures.

Warmer temperatures associated with climate change are expected to have a direct impact on plant pathogens, challenging crops and altering plant disease profiles in the future. In this study, we have investigated the effect of increasing temperature on the pathogenic fitness of Fusarium pseudograminearum, an important necrotrophic plant pathogen associated with crown rot disease of wheat in Australia. Eleven wheat lines with different levels of crown rot resistance were artificially inoculated with F. pseudograminearum and maintained at four diurnal temperatures 15/15°C, 20/15°C, 25/15°C and 28/15°C in a controlled glasshouse. To quantify the success of F. pseudograminearum three fitness measures, these being disease severity, pathogen biomass in stem base and flag leaf node, and deoxynivalenol (DON) in stem base and flag leaf node of mature plants were used. F. pseudograminearum showed superior overall fitness at 15/15°C, and this was reduced with increasing temperature. Pathogen fitness was significantly influenced by the level of crown rot resistance of wheat lines, but the influence of line declined with increasing temperature. Lines that exhibited superior crown rot resistance in the field were generally associated with reduced overall pathogen fitness. However, the relative performance of the wheat lines was dependent on the measure of pathogen fitness, and lines that were associated with one reduced measure of pathogen fitness did not always reduce another. There was a strong correlation between DON in stem base tissue and disease severity, but length of browning was not a good predictor of Fusarium biomass in the stem base. We report that a combination of host resistance and rising temperature will reduce pathogen fitness under increasing temperature, but further studies combining the effect of rising CO2 are essential for more realistic assessments.

Fusarium spp. are devastating fungal pathogens which cause significant losses in many cereal crops like wheat, maize, and barley. Genetic improvement of disease resistance requires an improved understanding of defense-associated processes operating in the host in response to an attack by Fusarium spp. Brachypodium distachyon is emerging as a model where host-cereal-infecting pathogen interactions can be studied conveniently. However, this requires developing an efficient infection assay that facilitates quick screening of germplasm (e.g., mutant lines). Here, we provide an efficient and reproducible Fusarium infection assay for Brachypodium. We believe this method will help further develop Brachypodium as a model for genetic improvement of disease resistance in cereals against Fusarium pathogens.

Fusarium spp. are devastating fungal pathogens which cause significant losses in many cereal crops like wheat, maize, and barley. Genetic improvement of disease resistance requires an improved understanding of defense-associated processes operating in the host in response to an attack by Fusarium spp. Brachypodium distachyon is emerging as a model where host-cereal-infecting pathogen interactions can be studied conveniently. However, this requires developing an efficient infection assay that facilitates quick screening of germplasm (e.g., mutant lines). Here, we provide an efficient and reproducible Fusarium infection assay for Brachypodium. We believe this method will help further develop Brachypodium as a model for genetic improvement of disease resistance in cereals against Fusarium pathogens.

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana‐infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole‐genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1–SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF‐1α/RPB1/RPB2 (translation elongation factor‐1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.

Ginger is considered by many people to be the outstanding member among 1400 other species in the family Zingiberaceae. Not only it is a valuable spice used by cooks throughout the world to impart unique flavour to their dishes but it also has a long track record in some Chinese and Indian cultures for treating common human ailments such as colds and headaches. Ginger has recently attracted considerable attention for its anti-inflammatory, antibacterial and antifungal properties. However, ginger as a crop is also susceptible to at least 24 different plant pathogens, including viruses, bacteria, fungi and nematodes. Of these, Pythium spp. (within the kingdom Stramenopila, phyllum Oomycota) are of most concern because various species can cause rotting and yield loss on ginger at any of the growth stages including during postharvest storage. Pythium gracile was the first species in the genus to be reported as a ginger pathogen, causing Pythium soft rot disease in India in 1907. Thereafter, numerous other Pythium spp. have been recorded from ginger growing regions throughout the world. Today, 15 Pythium species have been implicated as pathogens of the soft rot disease. Because accurate identification of a pathogen is the cornerstone of effective disease management programs, this review will focus on how to detect, identify and control Pythium spp. in general, with special emphasis on Pythium spp. associated with soft rot on ginger.