Biocontrol Potential of an Endophytic Pseudomonas poae Strain against the Grapevine Trunk Disease Pathogen Neofusicoccum luteum and Its Mechanism of Action.

Grapevine trunk diseases (GTDs) impact the sustainability of vineyards worldwide and management options are currently limited. Biological control agents (BCAs) may offer a viable alternative for disease control. With an aim to develop an effective biocontrol strategy against the GTD pathogen Neofusicoccum luteum, this study investigated the following: (1) the efficacy of the strains in suppressing the BD pathogen N. luteum in detached canes and potted vines; (2) the ability of a strain of Pseudomonas poae (BCA17) to colonize and persist within grapevine tissues; and (3) the mode of action of BCA17 to antagonize N. luteum. Co-inoculations of the antagonistic bacterial strains with N. luteum revealed that one strain of P. poae (BCA17) suppressed infection by 100% and 80% in detached canes and potted vines, respectively. Stem inoculations of a laboratory-generated rifampicin-resistant strain of BCA17 in potted vines (cv. Shiraz) indicated the bacterial strain could colonize and persist in the grapevine tissues, potentially providing some protection against GTDs for up to 6 months. The bioactive diffusible compounds secreted by BCA17 significantly reduced the spore germination and fungal biomass of N. luteum and the other representative GTD pathogens. Complementary analysis via MALDI-TOF revealed the presence of an unknown cyclic lipopeptide in the bioactive diffusible compounds, which was absent in a non-antagonistic strain of P. poae (JMN13), suggesting this novel lipopeptide may be responsible for the biocontrol activity of the BCA17. Our study provided evidence that P. poae BCA17 is a potential BCA to combat N. luteum, with a potential novel mode of action.

Persistence of atrazine and trifluralin in a clay loam soil undergoing different temperature and moisture conditions.

Dissipation kinetics of atrazine and trifluralin in a clay loam soil was investigated in a laboratory incubation experiment under different temperature and moisture conditions. The soil was spiked with diluted atrazine and trifluralin concentrations at 4.50 and 4.25 mg/kg soil, respectively, the moisture content adjusted to 40, 70, and 100% of field capacity (FC) and then incubated in three climatic chambers at 10, 20, and 30 °C. For each of the herbicides, soil samples were collected at 0, 7, 21, 42, 70, and 105 days and analysed by Gas Chromatography-Electron Capture Detector (GC-ECD). A stochastic gamma model was used to model the dissipation of herbicides from the clay loam soil by incorporating environmental factors as covariates to determine half-life and days to complete dissipation. Results showed that temperature played a greater role on atrazine persistence than soil moisture; while the interaction effect of temperature and moisture was significant on the persistence of trifluralin over time. Atrazine dissipated more rapidly at 30 °C compared to 10 and 20 °C, with a half-life of 7.50 days and 326.23 days to reach complete dissipation. Rapid loss of trifluralin was observed at 70% moisture content when incubated at 30 °C, with a half-life of 5.80 days and 182.01 days to complete dissipation. It was observed that the half-life of both herbicides tended to double with every 10 °C decreases of temperature over the range tested. The model indicated that both atrazine and trifluralin have the potential to persist in clay loam soil for several years at temperature ≤20 °C; which could potentially affect following crops in rotation.

Resistance in Maize (Zea mays) to Isolates of Puccinia sorghi from Eastern Australia.

The causal agent of maize common rust (CR), Puccinia sorghi, has increased in incidence and severity in Australia in recent years, prompting the assessment of sources of resistance and a preliminary survey of the diversity of P. sorghi populations. The maize commercial hybrids tested carried no resistance to 14 isolates of P. sorghi and had infection types comparable with that of a susceptible check. The resistance gene Rp1_D that remained effective in the United States for 35 years was ineffective against 7 of the 14 isolates. Maize lines carrying known "resistance to Puccinia" (Rp) genes were inoculated with the five isolates considered most diverse based on year of collection (2018 or 2019), location (Queensland or Victoria), and host from which they were isolated (maize or sweet corn). Lines carrying the resistance genes RpG, Rp5, Rp1_E, Rp1_I, Rp1_L, RpGDJ, RpGJF, and Rp5GCJ were resistant to all five isolates and to isolates collected in many agroecological regions. These lines were recommended as donors of effective resistance for maize breeding programs in Australia. Lines carrying no known resistance or resistance genes Rp8_A, Rp8_B, Rp1_J, Rp1_M, Rp7, and Rpp9 (conferring resistance to P. polysora) were susceptible to all five isolates. Differential lines carrying resistance genes Rp1_B, Rp1_C, Rp1_D, Rp1_F, Rp1_K, Rp3_D, or Rp4_A were either resistant or susceptible depending upon the isolate used, showing that the isolates varied in virulence for these genes. Urediniospore production was reduced on adult compared with juvenile plants, presumably due to changes in plant physiology associated with age or the presence of adult plant resistance.

Diversity Profiling of Grapevine Microbial Endosphere and Antagonistic Potential of Endophytic Pseudomonas Against Grapevine Trunk Diseases.

Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide. The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic endophytes, which may form varied relationships with the host plant from symbiotic to pathogenic. To explore the interaction between grapevine endophytic bacteria and GTDs, the endomicrobiome associated with grapevine wood was characterized using next-generation Illumina sequencing. Wood samples were collected from grapevine trunks with and without external symptoms of GTD (cankers) from two vineyards in the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of the endophytic community was conducted using the 16S rRNA gene (341F/806R) and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated the bacterial community in canker-free grapevine tissues in both locations, comprising 56-74% of the total bacterial population. In contrast, the Pseudomonas population in grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results suggest the potential of Pseudomonas species from grapevine wood to be used as biocontrol agents to manage certain GTD pathogens.

Syringopeptin Contributes to the Virulence of Pseudomonas fuscovaginae, Based on sypA Biosynthesis Mutant Analysis.

Pseudomonas fuscovaginae, first reported from Japan in 1976, is now present in many agroecological regions around the world; it causes sheath brown rot of rice and is reported as a pathogen of a broad range of hosts. The pathogen can infect rice plants at all stages of growth and is known to cause significant losses due to grain discoloration, poor spike emergence and panicle sterility. Limited information is available on the virulence and mechanisms of pathogenicity for P. fuscovaginae. To address this, an analysis of genomes was conducted, which identified the presence of a gene showing homology to one of the genes contributing to syringopeptin synthetase (sypA) of P. syringae pv. syringae. To study the potential role of this gene in the virulence and pathogenicity of P. fuscovaginae, a site-specific mutation was created. Following inoculation of seeds and plantlets of rice and wheat with P. fuscovaginae wild types and their respective mutants, we demonstrated that the mutation significantly reduced virulence. This was evident on rice and wheat inoculated with mutants causing a significantly higher number of roots, length of roots and seedling height compared with their respective wild types. Characteristic disease symptoms of necrotic lesions were significantly less in rice seedlings infected with bacterial suspensions of mutants indicating a reduction in virulence. Chromatography analysis of bacterial exudates showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants. These data demonstrate that the protein encoded by this sypA homolog gene is a major virulence determinant of P. fuscovaginae.

Draft Genome Sequences of Endophytic Pseudomonas spp. Isolated from Grapevine Tissue and Antagonistic to Grapevine Trunk Disease Pathogens.

Endophytic strains of Pseudomonas were isolated from grapevine tissues and exhibited antagonistic activity against several grapevine trunk disease pathogens. The draft genome sequences of the four strains revealed the presence of putative gene clusters that may impart biocontrol activity against plant pathogens.

Development of a Genomics-Based LAMP (Loop-Mediated Isothermal Amplification) Assay for Detection of Pseudomonas fuscovaginae from Rice.

The vast amount of data available through next-generation sequencing technology is facilitating the design of diagnostic marker systems. This study reports the use of draft genome sequences from the bacterial plant pathogen Pseudomonas fuscovaginae, the cause of sheath brown rot of rice, to describe the genetic diversity within a worldwide collection of strains representing the species. Based on a comparative analysis with the draft sequences, primers for a loop-mediated isothermal amplification (LAMP) assay were developed to identify P. fuscovaginae. The assay reported here reliably differentiated strains of P. fuscovaginae isolated from rice from a range of other bacteria that are commonly isolated from rice and other plants using a primer combination designated Pf8. The LAMP assay identified P. fuscovaginae purified DNA, live or heat-killed cells from pure cultures, and detected the bacterium in extracts or exudates from infected host plant material. The P. fuscovaginae LAMP assay is a suitable diagnostic tool for the glasshouse and laboratory and could be further developed for in-field surveys.

Genome-wide association analyses of common wheat (Triticum aestivum L.) germplasm identifies multiple loci for aluminium resistance.

Aluminium (Al3+) toxicity restricts productivity and profitability of wheat (Triticum aestivum L.) crops grown on acid soils worldwide. Continued gains will be obtained by identifying superior alleles and novel Al3+ resistance loci that can be incorporated into breeding programs. We used association mapping to identify genomic regions associated with Al3+ resistance using 1055 accessions of common wheat from different geographic regions of the world and 178 polymorphic diversity arrays technology (DArT) markers. Bayesian analyses based on genetic distance matrices classified these accessions into 12 subgroups. Genome-wide association analyses detected markers that were significantly associated with Al3+ resistance on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5B, 6A, 6B, 7A, and 7B. Some of these genomic regions correspond to previously identified loci for Al3+ resistance, whereas others appear to be novel. Among the markers targeting TaALMT1 (the major Al3+-resistance gene located on chromosome 4D), those that detected alleles in the promoter explained most of the phenotypic variance for Al3+ resistance, which is consistent with this region controlling the level of TaALMT1 expression. These results demonstrate that genome-wide association mapping cannot only confirm known Al3+-resistance loci, such as those on chromosomes 4D and 4B, but they also highlight the utility of this technique in identifying novel resistance loci.

Genetic characterization of a novel Phomopsis sp., a putative biocontrol agent for Carthamus lanatus.

A large number of isolates of Phomopsis sp. have been collected from the weed Carthamus lanatus (saffron thistle) in Australia, and their potential as biological control agents for weeds of the Asteraceae has been demonstrated. An analysis of their genetic diversity and a multigene phylogenetic analysis were undertaken to ascertain whether these isolates were distinct from other species of Phomopsis that commonly attack crop species in Australia. Minimal variation was found between the Phomopsis spp. isolated from saffron thistle, except two isolates that appeared to share identity with Diaporthe helianthii and P. viticola. Analysis of the selected isolates from saffron thistle with the nucleotide sequence of the partial ITS and tefl-alpha regions demonstrated that the sequences were distinct from all other species of Phomopsis so far described from crops in Australia. These findings provide strong support for the recognition of these isolates as a separate species of Phomopsis. The implications of these findings are discussed in relation to biological control of saffron thistle.

Analysis of TaALMT1 traces the transmission of aluminum resistance in cultivated common wheat (Triticum aestivum L.).

Allele diversities of four markers specific to intron three, exon four and promoter regions of the aluminum (Al) resistance gene of wheat (Triticum aestivum L.) TaALMT1 were compared in 179 common wheat cultivars used in international wheat breeding programs. In wheat cultivars released during the last 93 years, six different promoter types were identified on the basis of allele size. A previous study showed that Al resistance was not associated with a particular coding allele for TaALMT1 but was correlated with blocks of repeated sequence upstream of the coding sequence. We verified the linkage between these promoter alleles and Al resistance in three doubled haploid and one intercross populations segregating for Al resistance. Molecular and pedigree analysis suggest that Al resistance in modern wheat germplasm is derived from several independent sources. Analysis of a population of 278 landraces and subspecies of wheat showed that most of the promoter alleles associated with Al resistance pre-existed in Europe, the Middle East and Asia prior to dispersal of cultivated germplasm around the world. Furthermore, several new promoter alleles were identified among the landraces surveyed. The TaALMT1 promoter alleles found within the spelt wheats were consistent with the hypothesis that these spelts arose on several independent occasions from hybridisations between non-free-threshing tetraploid wheats and Al-resistant hexaploid bread wheats. The strong correlation between Al resistance and Al-stimulated malate efflux from the root apices of 49 diverse wheat genotypes examined was consistent with the previous finding that Al resistance in wheat is conditioned primarily by malate efflux. These results demonstrate that the markers based on intron, exon and promoter regions of TaALMT1 can trace the inheritance of the Al resistance locus within wheat pedigrees and track Al resistance in breeding programmes.

Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia.

A collection of isolates of Rhizoctonia solani anastomosis group (AG) 2 was examined for genetic diversity and pathogenicity. Anastomosis reactions classified the majority of isolates into the known subgroups of AG 2-1 and AG 2-2 but the classification of several isolates was ambiguous. Morphological characters were consistent with the species, with no discriminating characters existing between subgroups. Vertical PAGE of pectic enzymes enabled the separation of zymogram group (ZG) 5 and 6 within AG 2-1, but not the separation of ZG 4 and 10 within AG 2-2. PCR analysis using inter-simple sequence repeats (ISSR) and the intron-splice junction (ISJ) region supported the separation of ZG 5 and 6, while the AG 2-2 isolates were separated by geographic region. A comparison of distance matrices produced by the zymogram analysis and PCR indicated a strong correlation between the marker types. Pathogenicity studies suggested canola (Brassica napus) cultivars were most severely affected by AG 2-1, while cultivars of two species of medic (Medicago truncatula cv. Caliph and M. littoralis cv. Herald) were susceptible to both AG 2-1 and 2-2. The results indicate that AG 2 is a polyphyletic group in which the classification of subtypes is sometimes difficult. Further investigation of the population structure within Australia is required to determine the extent and origin of the observed diversity.