Evolution and detection of Fusarium oxysporum f. sp. cepae in onion in South Africa

[摘要] ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporumSchlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) hasbeen identified as the leading cause of harvest and storage losses. This pathogen is ofworld-wide importance and causes Fusarium basal rot of onions (Allium cepa),affecting all onion growth stages. No information is available on the evolution,genetic diversity, molecular detection and inoculum sources of the South AfricanFocep population.Similar to what is the case for South Africa, limited information is availableon Focep in other regions of the world. World-wide, four vegetative compatibilitygroups (VCGs) and two single-member VCGs (SMVs) have been identified amongtwo Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focepsuggested by VCG analyses was confirmed through molecular analyses of isolatesfrom a few countries. Only the mating type (MAT)1-1 idiomorph has been reportedfor Focep isolates from Welsh onion (Allium fistulosum).The development of sustainable management strategies of Focep is dependenton knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether highthroughput molecular methods can be developed for identifying the most virulent andwidespread Focep genotypes and (iii) the role of seedlings and seeds as primaryinoculum sources, and the Focep genotypes associated with these growth stages.Therefore, the three main aims of the current study were to investigate theaforementioned three aspects.In the first aim of the study, the genetic diversity and evolution of Focep wasinvestigated using a collection of 79 F. oxysporum isolates from South Africa (27Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCGanalyses revealed the presence of six VCGs, four among the Colorado Focep isolates(VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associatedisolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGsin Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible(HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongationfactor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. Thephylogeny divided the Focep isolates into two main clades, of which one containedthe two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. Thesecond, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, andnon-pathogenic isolates. Unlike the clade containing the two main VCGs, which werehighly virulent toward onion bulbs, the ancestral clade contained isolates that weremostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets withan intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphswithin the same isolate for some isolates, suggested possible exchange of geneticmaterial between isolates.The second aim of the study was to develop molecular methods for identifyingthe two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods andsequence-characterized amplified region (SCAR) markers. These techniques werefirst developed using the F. oxysporum isolates from the first aim, and were then usedto investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporumisolates from onion bulbs in South Africa. Two random amplified polymorphic DNAprimers provided two diagnostic amplicons for VCG 0425, but attempts to developSCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposonamplified polymorphism (IRAP) fingerprinting method enabled thedeveloped of a multiplex IR-SCAR polymerase chain reaction method that detectedthe VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCARmarker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in SouthAfrica associated with mature onion bulbs, since 63 of the Focep isolates had themolecular characteristics of VCG 0425.The third aim of the study was to determine whether seed and seedlingtransplants are inoculum sources of Focep, and whether the same genotype (VCG0425) that dominated on mature bulbs could be detected from these sources. Focepisolates were obtained from seven of the 13 investigated onion seed lots, as well asfrom onion seedling transplants that were collected from all five onion nurseries in theWestern Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but theseedborne nature of Focep was confirmed by showing that a green fluorescent proteinlabelled Focep transformant could be transmitted from infected soil to onion seed viathe onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings areinoculum sources of Focep. However, the Focep genotypes on seed and seedlings aredifferent from those in mature bulbs and were not dominated by VCG 0425.Furthermore, most (≤ 60%) of the seed and seedling isolates were moderatelyvirulent, as compared to the mostly highly virulent isolates from mature bulbs.

[发布机构] Stellenbosch University