A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species-specific PCR and DNA sequencing from a collection of 174 Ilyonectria-like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP-PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour-joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra- and inter-vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non-self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1-year-old potted vines showed variability in virulence; however, no correlations were detected.

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Isolates morphologically identified as Cylindrocladiella parva were isolated from characteristic black foot symptoms on a grapevine (Vitis vinifera) rooted on 101-14 rootstock from Central Otago in 2005 and 101-14 rootstocks from a nursery in the Auckland Region in 2007 and 2008. On potato dextrose agar, the isolates initially produced cottony, white mycelia that turned grayish cream or golden cream within 10 days, the initially tawny colony undersides becoming dark brown with age. Conidia (0 to 1 septate; 16.4 to 17.0 [16.7] × 2.3 to 2.6 [2.5] μm) and abundant chlamydospores were produced. To confirm identity of the isolates, genomic DNA was extracted and the ribosomal DNA (rDNA) and β-tubulin gene were amplified and sequenced (3,4). Sequences of the PCR products were compared with sequences in GenBank. The rDNA (535 bp) and β-tubulin (297 bp) sequences of the four isolates were 100 and 99% identical, respectively, to reported sequences of C. parva in GenBank (AY793454, grapevine isolate (4)/AY793455 for rDNA; AY793486/AY793488, grapevine isolate (4)/AY793489/HM034822 for β-tubulin). Although C. parva was previously isolated from grapevines in New Zealand (2) and rootstocks of mature grapevines, cuttings, and graft unions of grafted young grapevines in South Africa (4), its role as a pathogen of Vitis spp. has not been confirmed (2,4). However, it has been reported as a pathogen of Eucalyptus spp. (1) and was also isolated from Telopea speciosissima and Macadamia integrifolia in New Zealand (2,4). The C. parva isolates were tested as a mixed inoculum (four isolates) for pathogenicity on roots of 10 grapevine rootstock plants each of cvs. 101-14 and Schwarzmann (Sch). The rootstocks were grown in potting mix for 4 months, after which the root systems of all vines were wounded with an asparagus knife with a sharp, square tip, driven vertically down into the soil at four equidistant locations approximately 8 cm from the trunk. Each plant was inoculated with 50 ml of the mixed-isolate conidial suspension (106/ml), or 50 ml water (controls), followed by 50 ml of water. After 7 months of growth, the plants were harvested. For C. parva-inoculated plants, internal blackening of the stem base tissue was observed. Isolations from surface-sterilized trunk bases recovered C. parva from four and nine plants of 101-14 and Sch, respectively, with C. parva infections in 25 and 48%, respectively, of the four wood pieces taken per plant. Plants inoculated with water had no blackening and no C. parva was isolated from their stem bases. Mean shoot dry weights of inoculated plants (17.9 and 15.0 g for 101-14 and Sch, respectively) were significantly lower (P = 0.035) than noninoculated controls (26.5 and 20.0 g for 101-14 and Sch, respectively). Mean root dry weights were reduced by C. parva inoculation, although not significantly (32.7 and 27.0 g for C. parva inoculated 101-14 and Sch, respectively, and 36.2 and 27.4 g for control 101-14 and Sch, respectively). To our knowledge, this is the first report of C. parva as a pathogen of grapevines (2,4) and suggests that along with Cylindrocarpon spp., C. parva is part of the pathogen complex responsible for black foot of grapevines. References: (1) P. W. Crous et al. Plant Pathol. 42:302, 1993. (2) P. D. Gadgil et al. Fungi on Trees and Shrubs in New Zealand. Fungal Diversity Press, Hong Kong, 2005. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) G. J. van Coller et al. Australas. Plant Pathol. 34:489, 2005.

An in vitro method was developed to determine the relative pathogenicity of C macrodidymum C liriodendri and C destructans isolates on grapevine roots Young feeder roots of similar age and diameter were placed individually into Petri dishes containing watersaturated silica sand with the basal cut ends of the roots inserted into tubes filled with sterile water Three isolates each of C macrodidymum and C liriodendri and two of C destructans were inoculated as mycelial plugs onto the wounded roots three replicates per isolate After 1 month all inoculated roots had dark brown lesions (range of lengths 287404 mm) that were significantly different between isolates with an isolate of C liriodendri having the longest lesions and an isolate of C destructans the shortest This assay provides a rapid in vitro method to screen large numbers of Cylindrocarpon spp isolates