Genetic and biological characterisation of resistance to root lesion nematode Pratylenchus thornei in wheat.

Abstract

Root lesion nematodes of the genus Pratylenchus feed and reproduce in the root cortex of many plant species, including wheat. Migration through root tissue causes extensive root damage, and in turn severe reductions in growth and yield. In Australia, one of the most prevalent and widespread species affecting wheat is Pratylenchus thornei. Due to the wide host range of Pratylenchus spp. and the restrictions and inefficiency of chemical pesticides, the development of resistant cultivars has become increasingly important. Despite the identification and investigation of several resistance sources and resistance quantitative trait loci (QTL), no P. thornei resistance has been integrated into commercial cultivars. In addition, prior to this study, the biological resistance mechanisms of wheat against P. thornei were not well characterised. The identification of novel sources of genetic resistance in wheat and understanding of the biological mechanisms will allow effective combinations of genes either to be used alternatively or pyramided to generate effective and stable Pratylenchus resistance. The major objectives of the study were to identify genetic loci associated with P. thornei resistance and to investigate the associated biological mechanisms in a double haploid wheat population developed from a cross between the synthetically derived Sokoll and the Australian adapted Krichauff parental lines. The resistance to P. thornei observed in the Sokoll x Krichauff wheat population is complex and under the control of several loci which suppress all nematode developmental stages. The four main components of the root invasion process by Pratylenchus: root attraction, penetration, endoparasitic feeding and reproduction, were investigated to determine the location, timing and role of resistance against P. thornei. Through analysing root invasion by each nematode life stage, it was shown that resistance in the Sokoll x Krichauff population occurs post penetration to suppress P. thornei motility/migration and juvenile development causing reduced reproduction (egg deposition and hatch). Attraction and penetration assays were conducted on seedlings grown both in sand and on agar. There was no significant difference in the rate at which P. thornei was attracted towards resistant or susceptible roots in sand. However on agar, when both genotypes were present, there was a significantly higher attraction towards the susceptible roots indicating resistant roots may secrete repellent or toxic compounds during pre-penetration or that susceptible roots secrete more attractants. The penetration rates of P. thornei in resistant and susceptible roots, both on agar and in sand, did not significantly differ. No preferred root penetration zone was observed with P. thornei, but penetration was not random as nematodes were attracted to root regions previously invaded. In concordance with other Pratylenchus studies, resistance to P. thornei in this Sokoll x Krichauff population acts post penetration. Analysis of P. thornei development in the resistant and susceptible genotypes showed that significantly fewer P. thornei nematodes of all stages occurred in the resistant compared to the susceptible roots. Juvenile development was suppressed as no juvenile stage two nematodes (J2) were present 35 days after inoculation in resistant genotypes. At 45 days after inoculation, forty times more P. thornei juvenile stage three (J3) were present in the susceptible than the resistant parent. Unlike other studies where resistance against Pratylenchus caused nematodes to exit roots, in this study, similar numbers of P. thornei J2 were still present within the resistant roots 10 days after inoculation, indicating that resistance suppresses nematode development rather than causing nematodes to leave resistant roots. The inhibition of juvenile development resulted due to the suppression of nematode migration/motility which suppressed feeding but also due to reduced egg deposition and hatch. Simple and inexpensive assays were designed to investigate P. thornei motility, egg hatch and deposition in root exudates/extracts and roots grown on agar. Significantly higher numbers of P. thornei nematodes became non-motile when exposed to root exudates from resistant (65%) versus susceptible (30%) roots after exposure for 3 days. The effects of these compounds were found to be reversible and to specifically affect P. thornei but not Pratylenchus neglectus. In migration assays, P. thornei only migrated a small distance through the resistant root cortex from the point of inoculation (10 mm), but further in the susceptible roots (70 mm). Pratylenchus thornei reproduction was also affected by resistance. Egg deposition was up to 30% less within resistant than in the susceptible lines. About 40% less hatch occurred from eggs within and adjacent to roots of resistant versus susceptible seedlings. Similarly, hatching was decreased by 10% in resistant root exudate compared to the susceptible after 10 days of exposure. An increased hatch after dilution of root exudates and a lower hatch in resistant versus the absence of roots, indicates the presence of hatching inhibitor compounds. As these root exudates were derived from plants not exposed to Pratylenchus/other pathogens, this indicates resistant genotypes constitutively produce compounds that inhibit motility and reproduction. In order to identify QTL and develop molecular markers accounting for the observed resistance, a genetic map was constructed from the Sokoll x Krichauff doubled haploid population comprising 150 lines. A total of 860 Diversity Array Technology markers and 111 microsatellite markers were used to assemble the genetic map. Two highly significant P. thornei resistance QTL were identified on chromosomes 2BS and 6DS, QRlnt.sk-2B.1 and QRlnt.sk-6D, explaining 24 and 43% of the phenotypic variation, respectively. These QTL mapped to chromosome regions previously identified to be associated with Pratylenchus resistance, based on common marker locations. Two significant QTL were also identified on chromosomes 4A and 5A, explaining 6 and 9% of the phenotypic variation. The population was fixed for the effects of the highly significant QTL on 2BS and 6DS and further QTL were identified on chromosomes 2B, 2D, 3A, 5B and 6B. The QRlnt.sk-2B.1 and QRlnt.sk-6D account for a large portion of the observed resistance, showing that in this population the Sokoll derived resistance to P. thornei is very strong and is controlled by a few loci with large effects. There are considerable financial and labour costs associated with Pratylenchus phenotypic screening methods. Molecular markers employed through marker assisted selection will eliminate the need for large scale phenotyping in breeding programs and thus accelerate the development and availability of resistant cultivars. The microsatellite marker barc183 linked to QRlnt.sk-6D is also associated with P. thornei resistance in other mapping studies in different genetic backgrounds and thus highlights the potential benefit of this marker for use in marker assisted selection. However, the highly significant QTL on 2BS and 6DS currently span large chromosomal regions, thus fine mapping is required to delimit the QTL interval to establish more closely linked markers before they can be utilised in breeding programs. The ultimate aim of this project was to correlate a biological role with an identified P. thornei resistance QTL. Thus, in order to identify whether the QTL linked to P. thornei were associated with the observed motility and hatch inhibition, a subset of the population was phenotyped using the motility and hatching assays designed in this study. Suggestive QTL were identified on chromosomes 2B, 5B, 6B and 6D linked to hatching and motility suppression, which co-located to the P. thornei resistance QTL identified in this and previous studies. Although only suggestive, alignment with other QTL indicates that these resistance QTL may play a role in inhibiting P. thornei motility or juvenile hatching. To further define and confirm these QTL, phenotypic analysis needs to be performed on the entire population. The biochemical characteristics of the preformed resistant root compounds causing motility and hatching suppression were investigated. Root exudates that were subjected to heat/cold treatments caused less motility suppression than compared to the untreated control, indicating these resistant root compounds are water soluble and fairly stable in nature. Flavonoids, oxidised phenols and peroxidases associated with insect resistance genes that co-located with the hatching and motility suppression QTL and the P. thornei resistance QTL regions have been implicated in other Pratylenchus-plant resistance interactions. These results indicate a potential role for these compounds in the P. thornei resistance observed in Sokoll x Krichauff. Further investigation is required to define the chemical nature and specific roles of resistant root compounds in the suppression of nematode development. The results of this study show that the resistance observed in the Sokoll x Krichauff wheat population to P. thornei is complex and under the control of two highly significant and several minor loci, which do not affect penetration but suppress nematode feeding, development and reproduction.