Characterising sub-species variation in New Zealand Cylindrocarpon species that cause black foot of grapevines

Field: Agricultural and Biological Sciences (Life Sciences)

Black foot disease, caused by species of Cylindrocarpon, is a significant problem in New Zealand and throughout the world. From a 2005 study of symptomatic grapevine material contributed by 49 grape growers encompassing eight grape growing areas, 174 cultures of Cylindrocarpon-like isolates were recovered. The isolates were identified using a combination of morphological grouping, species specific PCR and sequencing of taxonomically informative genes. The collection contained five species of Cylindrocarpon, namely, C. liriodendri (57 isolates), C. destructans (53 isolates), C. macrodidymum (41 isolates),C. pauciseptatum (11 isolates) and Cylindrocarpon sp. (4 isolates). All three of the main Cylindrocarpon species reported worldwide were found throughout New Zealand, however, they differed in their distribution. A high proportion of the isolates recovered in the South Island were C. destructans (39%, n=46/118) as compared to the North Island (17%, n=8/48). A high proportion of the isolates recovered in the North Island were C. macrodidymum (45%, n=22/48) as compared to South Island (17%, n=20/118). The proportion of C. liriodendri isolates recovered from the North Island (27%, n=13/48) and from South Island (37%, n=44/118) were similar. Similar numbers of C. pauciseptatum and Cylindrocarpon sp. were isolated from both islands. The distribution of the three main species correlated with optimal temperature for growth where C. destructans had the lowest optima of 17.7°C and C. macrodidymum had the highest optima of 19.3°C (P<0.05). The data generated from UP-PCR analysis of the three main Cylindrocarpon species was used to create a neighbour joining tree which showed high inter- and intra-vineyard diversity, with few clonal isolates identified. The isolates in the neighbour joining trees formed four clades for C. liriodendri and C. destructans and six clades for C. macrodidymum. Nei’s measure of genetic diversity for the C. destructans, C. liriodendri and C. macrodidymum populations were H=0.3346, 0.1816 and 0.2580, respectively. Vegetative compatibility tests with seven C. destructans, nine C. liriodendri and eight C. macrodidymum isolates selected from different branches of the neighbour joining tree produced three VCGs for C.destructans and two VCGs for both C. liriodendri and C. macrodidymum. Only for C. destructans did pairing of isolates give rise to an interaction characterised as partially incompatible. Microscopic analysis of compatible reactions for the three species revealed the presence of hyphal anastomoses within and between the isolates and the formation of chlamydospores at the interaction zone of C. destructans isolates. Virulence varied between genetically different isolates of C. destructans, C. liriodendri and C.macrodidymum on detached root assay and potted vine assay. In the detached root assay 17, 15 and 14 isolates of C. liriodendri, C. destructans and C. macrodidymum were analysed; with isolate Wpa1c of C. liriodendri producing the largest lesion and isolate Ack2d of C. destructans producing the smallest lesion. There were differences in the appearance of lesions with C. macrodidymum producing more mascerated root tissue than the other two species. In the potted vine assay, the furthest recovery at 3 and 6 cm was from vines infected by isolates of C. macrodidymum. At 8 cm isolates of both C. liriodendri and C. macrodidymum were recovered in similar proportions. In general, the virulence showed by isolates of Cylindrocarpon species on detached roots correlated with their abilities to move up the stem bases in the potted vine assay. No relationship was found between genetic groups and virulence. Attempts to produce a transformation system for Cylindrocarpon isolates by Agrobacterium tumefaciens or protoplast mediated transformation were unsuccessful. Biochemical analysis using qualitative and quantitative assays showed that all three main species produced both laccase and acid protease in vitro. Among the three species, C. macrodidymum produced laccase activity that was up to 10 times greater than that of the other two species (P=0.000). For acid protease both C. destructans and C. macrodidymum produced greater activity than C. liriodendri (P=0.000). For both enzymes it was unclear whether greater activity was due to more enzyme or more active enzyme being produced. Using degenerate PCR the genes encoding lcc1 laccase and acid protease of the three Cylindrocarpon species were isolated. For the lcc1 gene 519 bp (93%) of the predicted 558 bp gene was isolated and this was translated into a 173 residue polypeptide. The predicted amino acids showed variation between species and isolates with variable enzyme activity. Of the 31 polymorphic residues identified six non-conservative changes produced between species and eight produced non-conservative changes in isolates; and only one of these (Δ86D→R) affected a residue at a site conserved in laccase enzymes. For the acid protease gene 280 bp (87%) of the predicted 319 bp coding sequence was amplified and sequenced. The 85 residue predicted amino acid sequence showed no variation between isolates with differing acid protease activity. Overall the study demonstrated substantial sub-species variation in genotype, virulence and enzyme production by species of Cylindrocarpon present in New Zealand vineyards.