Biological control of the grapevine trunk disease pathogens : pruning wound protection

[摘要] In recent years, several studies have conclusively shown that numerous pathogens,including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as wellas Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline anddieback of grapevines. These pathogens have the ability to infect grapevines through pruningwounds, which leads to a wide range of symptoms developing that includes stunted growth,cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16weeks after pruning and sustained levels of pruning wound protection is therefore required.The aims of this study were to (i) evaluate the ability of several biological agents to protectpruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes ofaction and (iii) determine the optimal time of season for biological agent application.Several biological agents were initially evaluated in a laboratory for their antagonismagainst trunk disease pathogens. The best performing control agents were tested in a fieldtrial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs werepruned to three buds and the fresh pruning wounds were treated with benomyl as a controltreatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillussubtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruningwounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp.(Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae),Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabasedproducts and isolates in most cases showed equal or better efficacy than benomyl,especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability tocolonise the wound tissue.The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which wereshown to be highly antagonistic toward the grapevine trunk disease pathogens, wereidentified by means of DNA comparison, and their ability to inhibit the mycelium growth ofthe trunk disease pathogens by means of volatile and non-volatile metabolite productionstudied. The two gene areas that were used include the internal transcribed spacers (ITS 1and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentagesimilarity determined and the two Trichoderma isolates were identified as Trichodermaatroviride. The volatile production of T. atroviride isolates was determined by placing aninverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealedwith parafilm. Trichoderma isolates were grown for 2 days on PDA where after they wereinverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% forL. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than forthe volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L.theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelialinhibition than USPP-T2.The timing of pruning wound treatment and subsequent penetration and colonisationof the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanccultivars were grown in a hydroponic system, pruned and spray treated with a sporesuspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. Onintervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissectedlongitudinally. From the one half, isolations were made at various distances from the pruningsurface, while the other half was observed under ultra-violet light to determine the depth offluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound,Trichoderma was isolated only from the wound surface and shallow depths into the wound (2to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6mm, which suggests that initial isolation of Trichoderma at these depths was resultant of thephysical deposition of conidia deeper into the pruning wound tissue, whereas the isolation ofTrichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In avineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz andChenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days afterpruning. One week after treatment, the distal nodes were removed and dissectedlongitudinally. Each half was observed under UV light and the pigment penetrationmeasured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeperpenetration of the pigment than pruning wounds treated in September. Moreover, pruningwounds made in September showed pigment particles in longitudinal sections up to 1 dayafter pruning, whereas wounds made in July showed pigment particles up to 3 days in thexylem vessels. These findings suggest that the best time for application of a biologicalcontrol agent should be within the first 24 hours after pruning.

[发布机构] Stellenbosch University