Phaeomoniella chlamydospora: potential inoculum sources in the grapevine propagation process

Abstract

Phaeomoniella chlamydospora is a recently described fungal pathogen that infects grapevines causing Petri disease, which is observed externally as a general decline. The objective of this research programme was to identify potential sources of inoculum in the grapevine propagation process (including propagation material, the grafting process and nursery beds) that may result in infected young vines.

Methodology for culturing and maintaining the fungus were investigated. The optimum temperature for growth of the fungus was 25°C (germination after 12 h, colony diameter and sporulation after 21 d were 77.1 %, 31 mm and 7.3x10⁵ conidia/mm², respectively). Effective long-term storage was achieved by placing conidial suspensions at -80°C (contamination and viability was 0 and 100% respectively, after 2 years). In four attempts to develop an inoculation technique, the best result achieved was 47% of vines infected. The occurrence of natural infections in control vines often masked treatment effects, this was overcome by the use of an isolate-specific genetic marker. Black streaking in vines, a symptom commonly used as a method of disease assessment, was shown to be prone to false positives, as the fungus was not isolated from 65% of positive samples.

Infected rootstock mother-vines were a source of inoculum as young own-rooted rootstock and grafted vines had infection levels of 42% and 18%, respectively, 8 months after planting. Infection increased over time but was absent in own-rooted scion until 20 months after planting. Vines from propagation material collected 0-600 mm from the mother-vine head had a higher level of infection 8 months after planting (42%) than those collected from further away (6-8%). By 20 months after planting, infection levels in vines from dormant propagation material collected close to the head and actively growing propagation material collected furtherest from the head were similar, 53 and 55%, respectively. The increase in infection levels observed over time may have been due to secondary infection from aerial conidia or changes in the physiological age of the vine, which affected either isolation or development of the pathogen.

A highly sensitive species-specific PCR-based detection method was used to detect P. chlamydospora DNA in samples collected from washings of propagation material, rehydration-fungicide tanks, pre-grafting rehydration tanks, washings of grafting tools and callusing media (13-50% of samples tested positive). The presence of live pathogen propagules was verified by traditional isolation methods. The PCR method was also used to detect aerial conidia in 10-30% of samples collected in a rootstock block and a grafting shed. When grafting was done in the absence of aerial conidia, the percentage of positives in wash samples of propagation material increased from 37% before to 70% after grafting indicating that the processing of infected propagation material could cause cross-contamination.

Phaeomoniella chlamydospora DNA was detected in 60% of soil samples collected from beneath known infected rootstock mother-vines using the species-specific PCR. As 17.5% of vines became infected when planted in artificially infested soil, a result verified using an isolate-specific genetic marker, it is possible that P. chlamydospora may be able to act as a soil-borne pathogen.

The results obtained have contributed to the understanding of potential inoculum sources of P. chlamydospora in the grapevine propagation process and have facilitated the development of best practice recommendations for propagators. These best practices are designed to prevent infection of propagation material in the field and reduce inoculum sources in the grafting process and nursery beds, by improved hygiene and crop rotation. The importance of understanding the influence of environmental factors (including temperature, relative humidity and rainfall), inoculum levels, the physiological age and status of the vine, and the presence of other pathogens on disease development has been highlighted. These issues still need further investigation and the molecular biology tools described in the current work will be of great benefit to such research.