School of Agriculture, Food and Ecosystem Sciences

Ascochyta rabiei (teleomorph: Didymella rabiei) which causes ascochyta blight is the most serious disease of chickpea (Cicer arietinum) in Australia as it causes significant losses in crop yield and quality. Although A. rabiei is heterothallic and genetically diverse elsewhere in the world, a study carried out on Australian isolates collected between 1995 and 2000 identified only one mating type and a low level of genetic diversity within the Australian A. rabiei population. In 2002, ascospores of Didymella rabiei, the sexual state of A. rabiei, were trapped in a discharge chamber, from chickpea stubble naturally infected with ascochyta blight in Western Australia. Examination of the stubble revealed pseudothecia typical of Didymella rabiei. The reported presence of the teleomorph in Western Australia indicated that the second mating type had been introduced into Australia or that the pathogen was capable of a low level of homothallic compatibility, previously undetected. The aims of this research were, to undertake a new survey of Australian A. rabiei isolates, to test for the presence of the second mating type, to determine if there has been a change in the diversity of the Australian population and to investigate if pathogenic variability was displayed among isolates. Sixty-seven isolates collected from chickpea fields in South Australia, New South Wales, Queensland, Victoria and Western Australia during the 2003 cropping season were single spored and confirmed as A. rabiei using a PCR test. The isolates were typed for mating type using MAT gene specific PCR primers and compared with tester isolates from USA. This test revealed that all the 67 isolates belonged to mating type 2 (MAT 1-2), thus, the presence of mating type 1 (MAT 1-1) in Australia could not be confirmed. Sequence Tagged Micro Satellite (STMS) markers were used to examine the genetic diversity of the A. rabiei isolates. The isolates were assessed for allelic variation at 19 microsatellite loci, each of which amplified a single locus. Seven of the loci were polymorphic across all the 67 isolates, while the remaining twelve were monomorphic. Based on the allele profiles at the seven polymorphic loci, 19 distinct A. rabiei haplotypes were identified with a total of 33 alleles. One haplotype constituted 35.8 % of the population and was found in the collections from South Australia, New South Wales, Queensland and Victoria. Cluster analysis did not show a clear distinction between isolates based on the state from which they were collected. Polymorphism across the 19 microsatellite loci revealed a slight elevation in diversity in the 2003-2004 population (Ht = 0.07; compared to 0.02 in the 1995 to 2000 collection) and an increase in the number of haplotypes compared with that detected in the previous study of Australian isolates. To examine the pathogenic variability of the Australian population of A. rabiei, nine isolates were inoculated on five chickpea differentials, ranging from highly susceptible to resistant, under controlled conditions optimal for A. rabiei growth and infection. Eight of the isolates were virulent on the susceptible and intermediate chickpea cultivars but not the resistant cultivar and one isolate was only virulent on the susceptible cultivar. Based on these results the isolates were classified into two pathotype groups. The results obtained from the study of the population structure and the pathogenic variability of A. rabiei in Australia will enable the Australian chickpea breeders to understand the A. rabiei population better for formulating management and breeding strategies.

The research program detailed in this thesis was initiated with industry support to study the process of leaf scorch, the effects of leaf scorch on William Bon Chretien (WBC) pear and if possible to develop practical damage thresholds for twospotted mite, Tetranychus urticae Koch, on WBC pear. At the beginning of this research program, leaf scorch regularly defoliated WBC pear trees in Australian orchards infested with twospotted mite. However, little was known about the process of leaf scorch, the factors involved with its development or importantly whether leaf scorch affected fruit yield. Leaf scorch of mite infested WBC pear trees had been observed by pear growers to occur rapidly especially on poorly irrigated trees. Leaf scorch was commonly thought to be caused by leaf desiccation resulting from either water loss from mite-punctured leaf surface or an upset in normal leaf function. As a result of these earlier observations the initial research focused on investigating the relationship between mite infestation and the development of leaf scorch and on monitoring the water relations of mite infested leaves. A glasshouse experiment was conducted to determine whether the water relations of WBC pear leaves were disrupted by mite feeding. The initial results did not confirm this, so other experiments were devised to further study the effects of mite feeding on the water relations of WBC pear leaves. The conclusion of these experiments was that twospotted mite did not affect the water potential or stomatal conductance of whole WBC pear leaves relative to uninfested leaves, even when leaf scorch was developing on the infested leaves. The effect of mite infestation on photosynthesis was also investigated, with only slight reductions recorded on mite-infested leaves. The glasshouse experiments did confirm that leaf scorch developed on mite-infested leaves only and that the level of leaf scorch damage was related to the level and possibly the duration of mite infestation. Reduced irrigation treatments confirmed that water stress significantly enhances the development of leaf scorch damage on mite-infested leaves. The level of leaf scorch on waterstressed trees was typically twice that of fully-irrigated trees, at given levels of mite infestation. This finding supported the earlier grower observations of a link between poor irrigation management and the development of leaf scorch damage and the study of the water relations of infested leaves. Leaf scorch failed to develop on any non mite-infested leaves, even in the reduced irrigation treatments where leaf water potentials were often lower than -2.5Mpa. This confirmed that there was a direct link between twospotted mite and leaf scorch and that a mechanism other than simple desiccation of the whole leaf was involved in the process of leaf scorch. Sensation is a red skinned pear cultivar that is closely related to the green skinned WBC pear. Pear growers had reported that the Sensation pear cultivar failed to develop leaf scorch when infested by twospotted mite, even though it was a closely related cultivar to WBC. Glasshouse experiments were also conducted to determine the differences in the response of the Sensation and WBC pear cultivars to twospotted mite infestation. The experiments concluded that Sensation is tolerant to twospotted mite, with no leaf scorch developing on mite infested Sensation leaves. The results also suggested that leaf scorch could possibly be developing on WBC pear as the result of a localised hypersensitivity response of WBC tissue to mite feeding. The effect of leaf scorch on flowering, fruit set, fruit size and fruit yield of WBC pear was monitored in commercial pear orchards. Late season or premature autumn flowering was observed on trees that were severely defoliated by leaf scorch damage in summer. However, flowering levels in the following spring were similar on all trees regardless of the level of leaf scorch damage that had developed in the previous season, even on those trees that had prematurely flowered in autumn. Fruit set was significantly reduced by up to 80 per cent on trees that were severely defoliated by leaf scorch in the previous season. Fruit size, which normally increases as fruit set decreases, was found to remain static on trees with low fruit set, even when only a few fruits remained on a tree. Fruit yield was significantly reduced on trees that were defoliated by leaf scorch in the previous season, as a direct result of reduced fruit set and the lack of fruit size compensation. The level of fruit set and yield were both responsive to the proportion of leaf area defoliated by leaf scorch in the previous season. This reduction in fruit set and yield was only observed in the year following defoliation and did not carry over to the second or third season. A field experiment was then established to determine if a relationship existed between twospotted mite infestation and the level of leaf scorch that develops on mite infested WBC leaves. The results obtained over two seasons clearly established that a combination of both the level and duration of mite infestation was strongly related to the level of leaf scorch that developed on WBC pear leaves. An index, Cumulative Leaf Infested Days or 'CLID', that combined both the duration of infestation and the proportion of leaves infested with twospotted mite gave the highest correlation with the development of leaf scorch damage on WBC trees. Irrigation treatments were also included in the experimental design and these confirmed that the development of leaf scorch was higher on water-stressed WBC trees compared with well-irrigated WBC trees. At this point, the research program had established in independent experiments both that leaf scorch damage occurred in a predictable response to an index of mite infestation and that the yield of WBC pear is reduced as the level of leaf scorch increases. The results from these separate experiments were then combined to develop preliminary damage thresholds for twospotted mite on WBC pear. The preliminary damage thresholds were set at 5 and 10 per cent of leaf area defoliated by leaf scorch which corresponds to 1500 and 2400 CLID per season, respectively. The preliminary thresholds were then field-tested in a number of commercial pear orchards over a period of three years. An improved mite monitoring protocol was also developed to enable commercial orchard scouts and pear growers to be involved in the field testing of the preliminary damage thresholds. In general, the level of leaf scorch damage that developed in the field testing stage was below or similar to the level that was expected by the CLID thresholds. In only one instance was the level of leaf scorch damage significantly higher than would have been expected given the level of CLID experienced by the trees in the block. An investigation into the irrigation and soil moisture records concluded that an unusually severe water stress had developed in this pear block and that this had enhanced the development of leaf scorch damage. In the first year of the field testing program, the majority of growers used their traditional calendar-based spray program for mite control. By the third year the majority of growers were using the damage thresholds to determine their spray program. This allowed them to optimise the benefit of mite predators, minimise chemical input and avoid significant yield loss as a result of leaf scorch damage. A computer program, 'MiteMaster', was developed for the last season of the field testing program. The Mitemaster program graphically displays the level of mite infestation, the level of beneficial predators of twospotted mite and the level of CLID calculated, and plots these against the damage thresholds. The scouts and growers indicated that the graphic display of data given by the Mitemaster computer program improved their understanding of the dynamics of mite control in their specific pear blocks. At the end of the field testing program the damage thresholds had been successfully validated under commercial conditions. A set of mite management guidelines, the damage thresholds and the updated MiteMaster computer program were released to the Australian pear industry.

School of Agriculture, Food and Ecosystem Sciences - Theses

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