N. L. Knight

An investigation of genetic variation among Australian isolates of Bipolaris sorokiniana from different cereal tissues and comparison of their abilities to cause spot blotch on barley

Bipolaris sorokiniana (teleomorph: Cochliobolus sativus), the causal agent of common rootrot (CRR) and foliar spot blotch (SB) diseases in barley and wheat, is an economically important fungal pathogen worldwide. However, the relationship between these two diseases is poorly understood. Differences within Australian B. sorokiniana populations were revealed by cluster analysis of amplified fragment length polymorphisms in genomic DNA of 48 B. sorokiniana isolates collected from the northern grain-growing region of Australia. Isolates collected from SB infections clustered apart from isolates collected from CRR infections. A subset of 31 B. sorokiniana isolates was assessed for their abilities to cause SB infections on barley leaves using a differential set of 15 barley genotypes and three other cereal species. The pathogen samples included 14 isolates from CRR infections of either wheat or barley and 14 isolates from SB infections of barley. Phenotypic experiments revealed that isolates of B. sorokiniana collected from barley SB infections showed a high level of pathogenic variability across the differential set. In contrast, isolates from CRR infections produced significantly less SB disease on inoculated barley leaves. Cluster analysis of the phenotypic infection response scores grouped isolates into three pathogenicity clusters demonstrating low, intermediate or high pathogenicity. The results of this study suggest divergence within Australian populations of B. sorokiniana in relation to host tissue specificity.

Assessment of Infection by Fusarium pseudograminearum in Wheat Seedling Tissues Using Quantitative PCR and a Visual Discoloration Scale

Assessment among cereal genotypes of relative seedling resistance to the crown rot pathogen Fusarium pseudograminearum has been primarily based on visual discoloration of the leaf sheaths. This study is the first to investigate the relationship between the widely used visual rating of seedling leaf sheath discoloration and the degree of colonization of these tissues by the pathogen, based on quantitative polymerase chain reaction (qPCR) of fungal DNA using primers specific for the translation elongation factor α sequence. Fourteen-day-old seedlings of four hard white spring wheat genotypes which differ in their degree of resistance to the pathogen, based on the expression of visible symptoms, were inoculated using a droplet method and assessed weekly from 7 to 35 days after inoculation (dai) for both discoloration and fungal DNA content per unit of tissue weight. Both visual assessment of disease symptoms and qPCR of fungal biomass indicated significant differences between the partially resistant and susceptible wheat genotypes from 14 dai. Visual discoloration of leaf sheath tissues was strongly correlated with fungal biomass estimated by qPCR in all four genotypes; however, this correlation became weaker with increasing time after inoculation. Significant correlations between these parameters were indicated at 14, 21, and 28 dai whereas, by 35 dai, the correlation was not significant. Evaluation of plants at 14 dai provided a rapid test which gave clear discrimination between lines for both parameters and was the time point of closest correlation between fungal colonization and disease symptoms. Symptom expression at all times following inoculation was accompanied by tissue infection, and at no time was symptomless infection observed under this screening environment. These qPCR results confirm that visual assessments of disease symptoms reflect the extent of tissue colonization by the pathogen in recently colonized tissues and confirm the validity of visual assessments for disease rating in high-throughput screening of breeding materials.

Histopathological assessment of Fusarium pseudograminearum colonization of cereal culms during crown rot infections

Histopathological assessment of the crown rot pathogen Fusarium pseudograminearum was performed using fluorescence microscopy of culm tissues of six cereal genotypes grown in inoculated field conditions. Tissue samples were collected at 10, 16, and 22 weeks after planting (WAP). Colonization of culm tissues was initiated through epidermal penetration, most distinctly through stomatal apertures, and progressed into the parenchymatous hypoderm, which exhibited the discoloration used as the basis for visual assessment of disease. Hyphae spread from the culm base vertically through the tissues, initially via the hypoderm and pith cavity. Colonization of sclerified cells occurred later in the disease process. Both xylem and phloem tissues became colonized by 16 WAP in all host genotypes, with colonization being less extensive in the more resistant genotypes. Culms displaying dead head symptoms revealed dense colonization in at least the first three internodes, with frequent xylem vessel and phloem cell occlusions. Paired living culms from the same plants exhibited less extensive colonization. These observations have revealed the ability of F. pseudograminearum to colonize all cell types of nodal and internodal sections, including vascular tissues, across all host genotypes. This study is the first detailed examination of the pattern of F. pseudograminearum colonization in adult hosts and indicates a potential vascular mechanism by which the effects of crown rot are produced.

Genome inheritance in populations derived from hexaploid/tetraploid and tetraploid/hexaploid wheat crosses

Hexaploid/tetraploid and tetraploid/hexaploid wheat hybrids were established using the hexaploid (Triticum aestivum L.) bread wheat LRC2010-150 and the tetraploid durum wheat (T. turgidum spp. durum) WID802. Thirty F2 progeny from each cross were characterised using Diversity Arrays Technology (DArTseq™) markers to determine whether there are differences between the crosses in the proportion of A, B and D genomic material inherited from each parent. Inheritance of the A and B genome from the tetraploid durum parent varied from 32 to 63% among the 60 lines assessed, and results indicated significant differences between the two F2 populations in the mean overall proportion of chromosomes A and B inherited from each parent. Significant differences were also observed between the crosses in the proportion of chromosomal segments on 2B, 3A, 3B and 4A inherited from the tetraploid parent. The F2 populations also showed significant differences in the average retention of D chromosomes per line with the tetraploid/hexaploid cross retaining a mean of 2.83 chromosomes while the reciprocal cross retained a mean of 1.8 chromosomes per line. A strong negative correlation was observed in individual lines from both populations between the proportion of the A and B genome inherited from the tetraploid durum parent and the retention of the D genome. The implication of these results for the design of efficient crossing strategies between hexaploid and tetraploid wheats is discussed.

Assessment of Fusarium pseudograminearum and F. culmorum Biomass in Seedlings of Potential Host Cereal Species

Fusarium crown rot is a major disease of wheat and barley worldwide, with the most frequently isolated causal agents being Fusarium pseudograminearum and F. culmorum. This study has successfully designed a quantitative polymerase chain reaction assay that is specific for F. culmorum, which has been used in conjunction with a previously established F. pseudograminearum-specific assay to compare the location and extent of infection by each fungus across a range of potential hosts, including six winter and three summer cereal species. All common winter cereals, excluding oat, demonstrated a similar range of visual and fungal biomass results when inoculated with either F. pseudograminearum or F. culmorum. Oat exhibited the lowest visual disease ratings and fungal biomass values of the winter cereals, while the sorghum, maize, and rice cultivars returned the lowest values overall. The ranking of host species according to visual discoloration was strongly correlated for both pathogens. Visual reactions to F. pseudograminearum were greater than those caused by F. culmorum in all potential hosts trialed; however, fungal biomass results only indicated this trend for barley. These results demonstrate significant variation in the ability of these pathogens to colonize the range of cereal species examined and also suggest differences between the pathogens in their patterns of host colonization.

Colonization of Durum Wheat (Triticum turgidum L. var. durum) Culms Exhibiting Premature Senescence (Dead Heads) Associated with Fusarium pseudograminearum Crown Rot

Fusarium crown rot is a significant disease of durum wheat (Triticum turgidum L. var. durum), which exhibits high levels of disease susceptibility. The most extreme symptom of crown rot is a prematurely senescing culm that typically fails to set grain. Individual crown rot-affected durum wheat plants displaying both nonsenescent and prematurely senescent culms were harvested to compare visual discoloration, Fusarium pseudograminearum biomass, and vascular colonization in culm sections sampled at three different heights above the crown. Field samples of EGA Bellaroi were collected at Wellcamp, QLD, in 2011, 2012, 2013, and 2014, and of Hyperno at Narrabri, NSW, in 2014. Prematurely senescent culms exhibited greater visual discoloration, F. pseudograminearum biomass, and vascular colonization than nonsenescent culms in each year they were examined. The extent of these differences varied between environments and timing of collection in each year. Vascular colonization initially occurred in xylem vessels and spread into phloem tissues as disease severity increased. The increased presence of hyphae in vascular bundles of prematurely senescing culms provides strong evidence for the hypothesis that restriction of water and nutrient movement in a diseased culm is a key factor in crown rot severity.