A Genetic Study on Resistance to Anthracnose Caused by Colletotrichum orbiculare in Cucurbits

概要

Anthracnose, caused by the fungal pathogen Colletotrichum orbiculare, is one of the most severe diseases in open-field cucurbits. Genetically conferred resistance is the most effective method to control anthracnose and the development of new cultivars with high resistance is eagerly anticipated. However, at present, there is limited knowledge, techniques, and breeding material in Japan to develop new resistant cultivars efficiently. In particular, searching for promising sources of resistance is vital and the identification of resistance genes is of great importance to construct DNA markers associated with resistance. In addition, clarifying the diversity of races and virulence in C. orbiculare distributed in Japan is also important for both breeding and cultivation. This study aimed to develop a basis for cucurbit breeding to improve anthracnose resistance in Japan.

　In Chapter 1, I summarize and discuss previous reports and studies on anthracnose in cucurbits. In Chapter 2, I screened 232 cucumber genetic resources of Asian origin and identified one accession, ‘Ban Kyuri’, with resistance to anthracnose. Further evaluation of the resistance based on seedling and greenhouse assays confirmed the resistance of ‘Ban Kyuri’. This Chapter also revealed variations in the virulence of three C. orbiculare strains in Japan. ‘Ban Kyuri’ exhibited resistance to all strains. Genotyping of CsSGR, a causal gene of cla, indicated that ‘Ban Kyuri’ is homozygous for the wild-type allele and its resistance level was equal to or higher than that of accessions harboring Cssgr, the mutated allele conferring resistance. This finding indicates that ‘Ban Kyuri’ harbors novel resistance genes. ‘Ban Kyuri’ cucumber is a promising source of resistance to C. orbiculare strains with diverse virulence in Japan.

　In Chapter 3, I focused on an old Japanese watermelon cultivar, ‘Tanso Teikosei’, with high resistance, preserved in the Nara Prefecture. I evaluated its resistance to diverse C. orbiculare strains distributed in Japan and revealed the inheritance of its resistance. ‘Tanso Teikosei’ and two other accessions were resistant to the highly virulent strain MAFF 306737. ‘Tanso Teikosei’ was also resistant to 16 other strains collected in Japan. Many strains caused severe symptoms in susceptible accessions, including the Japanese F1 cultivar. CAPS marker analysis indicated that NR28 and the two other resistant accessions were homozygous for the resistance allele of Cla001017 (causal gene for Ar-1). Susceptible accessions were homozygous for the susceptibility allele of Cla001017 (ar-1). Segregation analyses using progenies derived from crosses between ‘Tanso Teikosei’ and a susceptible accession suggested that Ar-1 caused the high resistance of ‘Tanso Teikosei’. In contrast, ‘Tanso Teikosei’ and other resistant accessions homozygous for Ar-1 were highly susceptible to the two strains sampled in Iwate Prefecture. This suggests that these strains have overcome Ar-1, similar to C. orbiculare race 2. ‘Tanso Teikosei’, the two other resistant accessions, and the CAPS marker designed in this study are promising materials for watermelon breeding for resistance to diverse C. orbiculare strains distributed in Japan that have similar pathogenicity as race 1.

　In Chapter 4, I examined the pathogenicity and virulence of 20 C. orbiculare strains in Japan to clarify the existence of races and virulence differences by cotyledon and seedling assays using four cucurbit host species: watermelon, cucumber, melon, and squash. Based on the symptoms on inoculated cotyledons and true leaves of watermelon, I evaluated the compatibility of C. orbiculare with each host cultivar and revealed the existence of three races in Japan based on the reaction to the host cultivar harboring the resistant gene Ar-1 (Cla001017). One of the most alarming results is that race 2, which overcomes Ar-1, an anthracnose-causing gene in current watermelon breeds in Japan, is also present in Japan and strains of race 2 may cause severe damage to watermelon production. In contrast, the cucumber and melon host cultivars showed quantitative and diverse symptoms regardless of the races defined by the watermelon resistance gene, whereas a squash cultivar was resistant to all strains. In particular, three strains caused severe damage even to the most resistant cucumber cultivar ‘Ban Kyuri’ and resistant cultivars harboring Cssgr, a gene conferring loss-of-susceptibility resistance. Using watermelon accessions harboring Ar-1 and ar-1 as the differential cultivars, breeders in Japan can determine the race of C. orbiculare. For cucumbers, ‘Ban Kyuri’ and accessions with Cssgr are desirable materials for evaluating the virulence level of C. orbiculare. Information on the races and diverse virulence in C. orbiculare helps to breed resistant cucurbit cultivars in Japan.

　In Chapter 5, I report genetic mapping of loci from this cultivar that confer resistance to MAFF 240422 and MAFF 306737 of C. orbiculare that belong to pathogenic races 0 and 1, respectively. Quantitative trait locus (QTL) analysis based on phenotypic data from 196 F2:3 families detected one major QTL, An5, and one minor QTL, An6.2, for resistance to race 0, and one major QTL, An2, and three minor QTLs (An1.1, An1.2 and An6.1) for resistance to race 1. I identified lysM domain receptor-like kinase 3 (CsaV3_5G036150) and wall-associated receptor kinase-like (CsaV3_6G048820) as candidate genes for An5 and An6.2, respectively. Multiple genes encoding pattern recognition receptors were located in the regions of the QTLs conferring resistance to race 1. Thus, I identified potential sources of genetic resistance to different pathogenic races of C. orbiculare in the Ban Kyuri cultivar of cucumber.

　In Chapter 6, I provide a comprehensive discussion on the development of anthracnose-resistant cucurbit cultivars based on the results of this study. The information on SNPs associated with each resistance QTL/gene examined in this study is a great tool, particularly for the utilization of anthracnose resistance sources in cucumber and watermelon breeding. This study also revealed the racial and virulence diversity of C. orbiculare strains distributed in Japan. Among them, some strains were considered to be invasive to cucurbit production. To control these invasive strains, pyramiding QTL/genes resistant to C. orbiculare provides robust resistance in cucumbers. This study also revealed the following challenges: Race 2 overcomes Ar-1 of watermelon and strains with strong virulence to cucumber cultivars with Cssgr and ‘Ban Kyuri’ are distributed in Japan. Therefore, the novel strain resistance from genetic resources must be screened in cucumber and watermelon. The information obtained in this study contributes to the development of new cultivars with anthracnose resistance in cucurbits, especially cucumber and watermelon.