Petri disease and esca are two of the most important grapevine trunk diseases that compromise the sustainability of viticulture worldwide. Petri disease is caused by Phaeomoniella chlamydospora and Phaeoacremonium species, while esca is caused by Fomitiporia species together with Petri disease fungi (Fig. 1). Other trunk disease pathogens, including Phomopsis species (Phomopsis dieback), Eutypa lata (Eutypa dieback) and species in the family Botryosphaeriaceae (Botryosphaeria dieback) (Fig. 1) are also associated with esca (White et al., 2011). These pathogens cause the decline and dieback of grapevines, which leads to severe economic losses due to the reduction in quantity and quality of grapes, as well as shorteningof the lifespan of grapevines.
The control of trunk disease pathogens is difficult, since no fungicides are registered for this purpose in South Africa. Consequently, the management of inoculum sources must form part of an integrated disease management strategy. Infected planting material is a known source of inoculum of Petri disease and esca pathogens, however, the mechanisms by which these pathogens spread from infected to healthy vines in vineyards remain unclear. Arthropods (animals with segmented bodies and legs, including spiders, scorpions, mites, millipedes, centipedes and insects) have been suspected of playing a role in the spread of these pathogens (Edwards et al., 2001; Van Niekerk et al., 2010), but little is known about the extent to which arthropods are associated with these pathogens. The aims of this research were to determine whether any of the arthropods occurring on declining grapevines are associated with trunk disease pathogens and with pruning wounds, and whether any of these arthropods could act as vectors of trunk disease pathogens.
Materials and methods
Arthropods were collected weekly in two vineyards (Stellenbosch and Rawsonville, South Africa) infected with grapevine trunk diseases for two years, using trunk and cordon traps (Fig. 2A), pruning wound traps (Fig. 2B), as well as visual inspections. Arthropods were rinsed with 2 ml of distilled water to collect fungal spores on their surfaces. DNA was extracted from 1 ml of these water samples and tested with specific primers to verify the presence of Phaeoacremonium spp. and Phaeomoniella chlamydospora. The remaining 1 ml was plated out on growth medium and fungal cultures thus obtained were identified by DNA sequencing.
Since Portuguese millipedes (Ommattoiulus moreleti) and cocktail ants (Crematogaster peringueyi) were found to be the most abundant arthropods on diseased grapevines, the potential of grapevine sap as a food source for these species was investigated in laboratory assays. Individual millipedes were offered a choice between water and grapevine sap (Fig. 3A), whilst ants in nests were presented with grapevine sap, tuna and water (Fig. 3B) and monitored for ingestion of sap. Subsequently, the ability of both groups to transmit a DsRedtransformed Phaeomoniella chlamydospora isolate to fresh pruning wounds on potted vines was tested. Millipedes and ants were confined on fungal cultures for 24 hours, transferred to the base of the plants and removed after three days. Faecal pellets of millipedes were also evaluated as potential sources of inoculum. Millipedes were fed on Phaeomoniella chlamydospora cultures for 24 hours, surface sterilised and allowed to defecate in sterile Petri dishes overnight. Faecal material was collected, macerated in water and plated onto potato dextrose agar.
FIGURE 1. Symptoms of major grapevine trunk diseases. A) Cross section of a Petri-diseased vine showing brown/black spots in the xylem vessels.
A total of 10 875 arthropod individuals, belonging to more than 31 families, were collected from declining grapevines (Moyo et al., 2014). The most abundant arthropods were millipedes, ants, spiders and beetles. Visual observations showed that Portuguese millipedes and cocktail ants were closely associated with fresh grapevine pruning wounds (Fig. 4). 33% of the 5 677 water samples analysed, contained spores of grapevine trunk disease pathogens. Of these, 37% were recovered from Portuguese millipedes, 22% from cocktail ants, 15% from spiders, 10% from beetles and the remaining 16% from various arthropods, such as springtails, bugs and earwigs. Spores of all the major groups of grapevine trunk diseases were detected on the arthropods. Phaeoacremonium species were detected in 1 242 samples, while Phaeomoniella chlamydospora was identified from 855 samples. Other fungi isolated included members of the Botryosphaeriaceae, Diatrypaceae and Diaporthales.
Results from the feeding experiments showed that both millipedes and ants preferred grapevine sap over the other food items, indicating close association with pruning wounds. A month after exposure to millipedes and ants, isolations from pruned shoots of potted grapevines revealed that Phaeomoniella chlamydospora was transferred onto pruning wounds and caused infection. Spores of Phaeomoniella chlamydospora survived passage through the gut of millipedes and viable spores present in their feaces were able to form colonies of Phaeomoniella chlamydospora.
A wide variety of arthropods were shown to be associated with grapevines infected with trunk diseases. Portuguese millipedes and cocktail ants were the most abundant of the species from which fungal spores were recovered. They are highly attracted to fresh pruning wounds, where they feed on the plant sap. It was shown that they are able to transfer spores of Phaeomoniella chlamydospora to infect grapevine pruning wounds while feeding on pruning wounds. It is highly likely that other grapevine trunk disease pathogens are transmitted in the same manner. Millipedes and ants are opportunistic or incidental vectors of trunk diseases, as they acquire the spores by coming into contact with the fruiting bodies of the fungi when spores are released or by feeding on the sap of diseased vines.
Fruiting bodies of Petri disease fungi occur within cracks and crevices of diseased grapevines, as well as on surfaces of old pruning wounds (Edwards & Pascoe, 2001; Baloyi et al., 2013). The fruiting bodies of Phaeoacremonium species have long necks which are often oriented towards the openings of cracks, pruning wounds and insect tunnels (Fig. 5). The cracks and crevices of diseased vines were observed to be prime habitats for several arthropods in this study (Fig. 6). The presence of fruiting bodies inside cracks and insect tunnels in grapevines supports an association between arthropods and Petri disease pathogens and enhances transmission of spores by arthropods.
It is evident that effective management of trunk diseases in vineyards can only be achieved by preventing the spread of fungal spores by these vectors. This can be done by preventing them from gaining access to pruning wounds by placing barriers or toxic baits at the base of the plants, or by protecting pruning wounds with chemical or biological control methods (Halleen et al., 2010). Removal of dead and diseased wood will not only reduce inoculum load, it will also help reduce arthropod populations, as dead wood is a prime habitat for them. Several ant control methods are available in South Africa and these can be applied in vineyards where ants have been identified as a problem. The control of ants, especially in older vineyards with visible Petri disease and esca symptoms, will in turn help to curb the spread of grapevine trunk disease pathogens.
At present we do not know whether millipedes feeding on grapevine buds and leaves cause economic damage. Given that they have been identified as vectors of trunk disease pathogens, further study in this regard is recommended before the application of control measures for millipedes can be justified. Further studies should also determine whether other arthropods (like spiders), which were also found to carry grapevine trunk disease pathogens, are involved in the transmission of these pathogens to fresh pruning wounds.
We acknowledge financial support from Winetech (Project WW06/39), NRF, THRIP and ARC; technical assistance from the Plant Protection Division (ARC Infruitec-Nietvoorbij) and Arachnology unit of the ARC Plant Protection Research Institute for identification of spiders.
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