We examined the genetic relatedness of isolates of Phaeomoniella chlamydospora, which is one of the most aggressive and common causal fungi of the trunk disease Esca (aka Measles). This same pathogen causes Petri disease (aka Young Vine Decline), which affects young vines, whereas Esca affects mature vines. It is important to know if isolates of the pathogen from different vineyards in different states represent one or a few clones, or if each isolate is a different genotype. Such patterns reflect the type of spore, asexual (conidia) or sexual (ascospores), respectively, by which the pathogen spreads. Different spore types have different capacities for dispersal, different consequences for fungicide resistance, and so knowing the spore type can inform disease management guidelines. The pathogen is presumably asexual. This is in part because no one has found the fruiting bodies that produce ascospores (perithecia); the fungus produces only conidia in culture. As such, Esca is thought to be spread by conidia. We used a population genetics approach to evaluate genetic relatedness of populations from California (58 isolates), British Columbia (12 isolates), and the northeastern US (26 isolates). Isolates were genotyped with 18 microsatellite markers. We performed a genetic clustering analysis based on their genetic similarity. Genetic diversity indices were computed for each cluster of isolates. Genetic differentiation among clusters was also estimated. Three genetic clusters were identified, with two clusters consisting of isolates from British Columbia and California, and the 3rd cluster of isolates from the northeastern US. All isolates had a different genotype, suggesting that the pathogen spreads by sexual spores. High genetic subdivision (Fst = 0.28; P < 0.001) suggests that genetic and/or ecological factors may maintain genetic differentiation among these three groups. Our findings indicate a ‘hotspot’ of genetic diversity in the northeastern US, which could correspond to a center of origin of the pathogen. Genetic diversity indices were also higher than in a previous study using the same set of microsatellite markers to compare European and Australian isolates. If the northeastern US is a center of origin of the pathogen, sources of disease resistance may be present in native Vitis species. Sexual reproduction may occur on hosts other than grapevine; this could explain the absence of perithecia from vineyards.
Grapevine leafroll disease is caused by grapevine leafroll associated viruses (GLRaVs). Several GLRaVs have been shown to be transmitted from vine to vine by mealybugs. Within this virus complex, GLRaV-3 is the most predominant species in the world. The invasion of the vine mealybug (Planococcus ficus) in California may result in increased disease incidence of established GLRaV-3 throughout the state. We studied characteristics of GLRaV-3 transmission [effect of acquisition and inoculation access period (AAP and IAP, respectively), latent period, virus retention, and persistence of infectivity] by the vine mealybug. Our results indicate that the vine mealybug transmits GLRaV-3 in a semi-persistent manner. First instar mealybugs were more efficient vectors than adult mealybugs. Virus transmission occurred with a 1-hour AAP and peaked with a 24-hour AAP. Vine mealybugs inoculated GLRaV-3 with a 1-hour IAP, and transmission efficiency increased with longer plant access period up to 24 hours, after which transmission rate remained constant. In addition, vine mealybugs transmitted GLRaV-3 with a short latent period. After an AAP of 48 hours, vine mealybugs lost GLRaV-3 and infectivity four days post-acquisition feeding. In summary, we systematically analyzed the transmission parameters of GLRaV-3 by the vine mealybug. This information will be valuable for the development of leafroll disease management practices.
Esca and Petri disease have been documented in all of the major viticulture production regions of California. Diseases caused by the vascular pathogen, Phaeomoniella chlamydospora, is more likely responsible for Petri disease, but esca can be caused by both P. chlamydospora and numerous species of Phaeoacremonium. These fungi are also responsible for poor vineyard establishment in many newly-planted vineyards. In these cases, the young vines may have been infected prior to planting, but it is not known from whence the pathogen comes. The infection courts for these fungi are generally the xylem parenchyma and vessels of mature grapevine xylem and we suspect that nursery infection is occurring through these structures. It is suspected that the pathogens may be passed from mother vines to progeny vines via spores carried either in the sap flow or by external contamination of bark by the release of ascospores from perithecia.
Currently, it is known that these fungi are present in propagation material coming from mother plants in nurseries. A detection method using nested-PCR was developed to provide a rapid and sensitive test to determine the presence of these fungi in grapevine material throughout the propagation process.
It was demonstrated that pruning wounds are susceptible to infection by conidia of both T. minima and P. chlamydospora. Spores of several Phaeoacremonium species and P. chlamydospora were trapped in infected vineyards. Also propagules of both fungi were found on the surface of clusters, leaves and trunks of grapevine in infected vineyards in California. Perithecia of T. minima have been identified on moist incubated infected grapevine woods in California and Australia. Natural perithecia were also found in the deep cracks and wounds in infected vineyards throughout California.
Two other species which have been reported only once before on grapevines in California, Pm. angustius and Pm. Mortoniae, were also found to be somewhat common in California. After confirming the presence of Togninia spp. and Pa. chlamydospora in nursery propagation wood and as overwintering structures in California vineyards, we have begun to examine different applications for disease management. Liquid lime sulfur has been shown to be effective in killing the spores inside the pycnidia of Pa chlamydospora and the ascospores inside the perithecia of T. minima. Additional dormant applications need to be conducted in the next few years to determine the effect of lime sulfur and other tested fungicides on disease expression in infected vineyards. This work will require long term studies to take into account the discontinuity of the disease. Our results indicated that pruning wounds were susceptible to several fungal pathogens responsible for shoot dieback of grapevine, and we also have recovered several of these fungi in concert from wood cankers. Therefore, we are now testing the ability of boron-based material in comparison to other fungicides to control several wood decay fungi of grapes including E. lata, Botryosphaeria rhodina, B. dothidea, B. obtusa, B. sarmentorum, Phaeomoniella chlamdospora, Phaeoacremonium aleophilum and Pleurostomophora richardsiae
Phaeoacremonium inflatipes, P. aleophilum, and Phaeomoniella chlamydospora are three recently described fungi that are involved in the development of symptoms of young vine decline and measles. Up to the present time it is not clear whether the fungi are already present in the propagation material, in mother plants, in scion or rootstock or in rooted cuttings which become infected during preparation, A detection method using nested-PCR is being developed to provide a rapid, sensitive, and specific test to determine the presence of these fungi in grapevine propagation materials.
Field experiments are being conducted to explore the epidemiology and biology of the young vine decline organisms and measles. P. Inflatipes and P. aleophilum are suspected to produce aerial fruiting structures and P. chlamydospora is known to produce pycnidia, microslerotia, and chlamydospora; however, the mode of dissemination of these fruiting structures is unclear. Spore traps were placed in select vineyards in northern and southern California where measles and young vine decline are known to occur. Initial results show that the 3 pathogens could be air borne fungi. This study will elucidate how the fungi spread and under what condition the spores are released to re-infect plants.
Our previous results showed that fresh wounds sustained by the plant at any time during the year, from pruning or other injuries, could provide suitable infection court for the pathogen. Also, that the 3 organisms are capable of infecting old wounds (4 months or older) and invading adjacent green tissues. The experiments were repeated this year to confirm these results.
Cuttings of 20 rootstocks were inoculated with measles and young vine decline organisms and allowed to callus and root following standard nursery procedures. After more than 1 year of growth (in pots inside the lath house), they are currently being evaluated for length of vascular streaking and %recovery of the pathogen. This study was conducted to determine differences in susceptibility of the rootstocks to measles and young vine decline organisms.
Development of a selective medium to facilitate isolation and recovery of the organisms causing measles and young vine decline, in infected tissue, spore traps and soil, is in progress. This will be particularly useful in soil isolation.
Greenhouse and field experiments are being conducted to determine the relation between levels of boron and susceptibility to measles and young vine decline. We are also presently looking at soil, plant, and water analyses from areas that have high incidences of the disease to see any relationship.
Pathogenicity trials have been done with Thompson Seedless, Flame Seedless, and Cabernet varieties inoculated with spore suspension of two species of Phaeoacremonium and Phaeomoniella chlamydospora (Phaeoacremonium chlamydosporum). Additional experiments are also underway to stimulate early fruit production on certain varieties of grape to study the measles symptoms on the berries. Spore traps have been placed in ten productions locations select vineyards where measles have occurred. Lab procedures have been developed to facilitate the recovery of Phaeoacremonium and Phaeomoniella spores from these traps. In vitro methods are being used to screen materials that may be effective against these pathogens.
Nine vineyards in California, including four table and five wine grape, were surveyed for measles throughout the 1997 growing season. Ratings of fruit and foliar symptoms were recorded for an acre of grapevines at each site in order to make a correlation between incidence and severity of measles and environmental conditions (data pending). Two healthy and two infected grapevines from each vineyard were selected in order to compare weights and soluble solids of the fruit. At all the sites the healthy fruit weighed more than infected fruit. Brix readings taken of healthy grapes were also higher at most sites except Kern and Tulare-1 where infected grapes were 0.7 and 1.0 brix reading higher than healthy grapes respectively. Entire grapevines were excavated and isolated from in order to determine the exact locations on the vine where Phaeoacremonium spp. reside. P. chlamydosporum was isolated from cordons, trunks and roots and P. inflatipes was isolated from cordons and trunks. Pathogenicity test using P. inflatipes and P. chlamydosporum resulted in 70.8%and 66.7%mortality of grape seedlings respectively. Inoculations of grape wood cuttings with P. inflatipes resulted in over 60%recovery of the fungus from necrotic tissue. Fugicides commonly used in vineyards were tested for growth inhibition of Phaeoacremonium spp. with Benlate and Procure limiting fungal growth most effectively after 7 days. Bayleton, Rally, Rubigan and sodium arsenite had little effect on the growth of P. inflatipes but growth inhibition was more significant against P. chlamydosporum.
Isolations from symptomatic grapevines have consistently yielded several fungi including Cladosporium, Penicillium, Alternaria, Rhizopus, Paecilomyces, Aspergillus, Phomopsis, Eutypa, Botryodiplodia, Scytalidium, Phaeoacremonium as well as others. Several of these fungi are known pathogens including Phomopsis, Eutypa, Botryodiplodia, Scytalidium, Cylindrocarpon, and Phaeoacremonium. Symptom expression is well documented for Eutypa and Botryodiplodia. Little is known about the about Phaeoacremonium, Scytalidium, and Cylindrocarpon and the disease symptoms they cause. Pathogenicity tests using grape seedlings and cuttings has shown Phaeoacremonium spp. to be capable of growing in the xylem of grapevines and results in stunting (of both foliage and root system), leaf distortion and discoloration. As of yet no fruit symptoms have resulted from the inoculations but a method for inducing fruit production on young grape cuttings is being utilized in order to obtain fruit symptoms. Pathogenicity test using Scytalidium and Cylindrocarpon have not resulted in symptom expression and plants continue to be monitored. Biology and epidemiology studies of Phaeoacremonium spp. are underway to determine aspects of life cycle and effects of environmental parameters. Vineyards with a history of measles are being surveyed in Santa Barbara, San Luis Obispo, Monterey and El Dorado counties. Vineyards are being monitored several times throughout the year so a disease curve can be developed. The curve will be plotted against environmental conditions (temperature, RH, etc.) in order to correlate incidence of measles with the environment. Severity of the disease is also being determined by rating each vine for foliar and fruit infection and determine crop loss. All weed species and wood cuttings left in the vineyard are being investigated as potential host and inoculum sources and their possible role in the life cycle of Phaeoacremonium. Several types of media are being tested for selectivity to Phaeoacremonium spp. in order to detect inoculum sources in soil and bark. Media are being amended with chemicals such as Benlate, PCNB, and Rose Bengal which inhibit the growth of competing fungi. Disease control is the primary component of this work. Chemicals are being screened against Phaeoacremonium, Scytalidium, and Cylindrocarpon. Benlate and related compounds are effective against Phaeoacremonium.