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Identifying Pestalotiopsis in the Vineyard and the Lab
Common fungus found to be causing disease in eastern vineyards
Species of the fungal genus Pestalotiopsis are present on grapevines in vineyards east of the Rockies, but growers didn’t realize it was the culprit behind some of their problems with foliage blight, berry rot, stem necrosis and trunk disease.
It was not until 2012 that J.R. Urbez-Torres, a plant pathologist from Agri-Food Canada, and his associates reported the pathogen’s association with vascular symptoms of trunk diseases and isolated it from symptomatic grapevines that exhibited characteristic dieback patterns. Their article, “Characterization of Fungal Pathogens Associated with Grapevine Trunk Diseases in Arkansas and Missouri,” identified Pestalotiopsis as the second-most-prevalent fungus in vineyards in those two states.
During the past several years, Dr. Dean S. Volenberg, viticulture and winery operations extension specialist at the Grape and Wine Institute of the University of Missouri, and Lucie Morton, a well-known viticultural consultant based in Charlottesville, Va., each found inexplicable symptoms on grapevines, leaves and fruit.
Volenberg identified the fungus Pestalotiopsis in Norton berry clusters and also in a canker on a Norton vine (which caused trunk die-back) and began writing about his findings in his weekly newsletter Vinews (Viticulture Information News) in 2015. Morton first found Pestalotiopsis on Maryland grapevines in 2009, and when she intensified her investigation of the fungus in 2016, she found Volenberg’s newsletters online.
The two researchers began to work together as they realized that Pestalotiopsis was causing problems from Missouri to states on the East Coast. They displayed two posters summarizing their evidence about the identity and prevalence of Pestalotiopsis at the American Society for Enology and Viticulture-Eastern Section meeting in Charlottesville, Va., this July.
Symptoms of Pestalotiopsis
Since 2015, Volenberg and Morton have identified foliar, berry and cluster symptoms (in the absence of grapevine dieback) on both Vitis vinifera and interspecific hybrid grapevines that seem to be caused by Pestalotiopsis.
In the Midwest, Volenberg first found the fungus in a Norton vine with trunk disease in June 2015. A few weeks later, he saw rot on clusters that he confirmed was caused by Pestalotiopsis. The following year, he received reports early in the season of leaf discoloration, leaves with brown spots or black smudges and leaves falling off 12- to 14-inch shoots. Upon testing, these problems were shown to be the result of Pestalotiopsis. As temperatures rose during the summer months in Missouri, there were fewer reports of problems created by the fungus.
On the East Coast, on the other hand, Morton found that the pathogen appeared to be causing the symptoms of berry shrivel and cluster stem wilt later in the season, after véraison. Pestalotiopsis also has been associated with rachis infections that result in berry shrivel in vinifera vineyards.
Background on Pestalotiopsis
The genus Pestalotiopsis consists of 205 described species. These species are differentiated based on conidial characteristics including size, septation, pigmentation and the presence or absence of apical appendages. The five-celled conidia have distinctive basal appendages that make them appear like microscopic insects. Pestalotiopsis is a complex genus and can be difficult to classify to the species level because characteristics such as growth rate, conidial morphology and fruiting structure tend to vary within species.
Pestalotiopsis species are considered to be weak pathogens, phytopathogens, saprophytes or endophytic symbionts. As a plant pathogen, Pestalotiopsis is thought to be opportunistic and can affect plants under stress. Many of the Pestalotiopsis species reported to be plant disease pathogens are not host specific, and this lack of host specificity has resulted in confusion in identifying and classifying species.
The range of symptoms that have been associated with Pestalotiopsis among grape species and within interspecific grape cultivars makes it difficult to diagnose the pathogen within a field setting. Volenberg and Morton have developed techniques to culture Pestalotiopsis both in-vivo and in-vitro for identification. Symptomatic grapevine tissue was collected and transported to laboratories located in Columbia, Mo., or Charlottesville, Va. They also took photographs to document the symptomology viewed in the field and in the laboratories.
In-vivo symptomatic tissue incubation
Samples were initially inspected for fungal fruiting structures, prior to moist incubation. Berry or plant tissue with the symptomology of Pestalotiopsis was moist incubated in sealed containers at 21° to 24° C, with the samples elevated above moist paper towels. The samples were under a 12-hour photoperiod supplied by fluorescent lights.
Conidiophores arising from the tissues were gently crushed in sterile water, and conidia were viewed with optical microscopy.
Black pycnidia producing black cirrhi (a mucus-bound ribbon-like mass of spores that exudes from a fungus) were observed on symptomatic leaf, petiole, pedicel and rachis tissue after 24 to 168 hours of incubation. Acervuli (small asexual fruiting bodies resembling a cushion or blister consisting of a mat of branching filaments that is produced on a host by some fungi) were only observed forming on grape berries after seven to 10 days of moist incubation. Characteristic five-celled conidia with apical and basal appendages were present in both cirrhi and acervuli.
In-vitro culture
Conidia from some samples were aseptically transferred within a laminar flow hood to full strength potato dextrose agar (PDA) in Petri plates to obtain pure cultures for species identification. Sealed Petri plates were placed under fluorescent lighting that provided a 12-hour photoperiod at 21° to 24° C. After 10 days, a 5-mm mycelium plug was transferred to Petri plates containing PDA and autoclaved carnation leaves or PDA, and the Petri plates were placed u
nder the conditions described above. The number of days until acervuli formed was noted.
The colonies of Pestalotiopsis on PDA were whitish with crenated edges and aerial mycelium. Acervuli formed after 20 days on PDA, whereas acervuli formed within 10 days when autoclaved carnation leaves were added to the PDA.
Two species of Pestalotiopsis have been identified from sequence data of the internal transcribed spacer (ITS)-rDNA, which was compared to GenBank sequences. Both Pestalotiopsis sydowiana and Pestalotiopsis microspore were identified.
Fungicide evaluation
Both Volenberg and Morton had realized early in their investigations into the symptoms of what turned out to be Pestalotiopsis that growers had changed their spray programs before their vines exhibited the new signs of fungal disease. Morton knew in 2014 that the grower had stopped using Pristine, a commonly used strobilurin-boscalid product, earlier in the season because he didn’t think they needed “strobies” in their spray program.
When Volenberg first saw rot on clusters after considerable rainfall in 2015, he reviewed the grower’s spray program and suggested he include Pristine. After it was applied, the affected berries dried up and dropped off. Subsequently, he confirmed the rot was caused by Pestalotiopsis. In Volenberg’s mid-June newsletter in 2016, he recommended that because Pestalotiopsis can cause fruit rots in addition to trunk and leaf problems, growers should include strobilurins such as Pristine, Abound and Flint, as well as mancozeb in their spray programs.
Based on their experiences with these fungicides, the two researchers decided to evaluate the efficacy of mancozeb and pyraclostrobin on Pestalotiopsis. Solutions of mancozeb and pyraclostrobin were added to PDA, and media was poured into Petri plates. Mancozeb and pyraclostrobin were evaluated at zero, 0.01, and 0.001 the recommended (mancozeb 3.36 kg/ha and Pristine 875 g/ha) field-use rate. Mycelium cores (5 mm) were placed in the center of each Petri plate. The Petri plates were evaluated for mycelium growth eight days after the mycelium transfer.
The results indicated the fungicides substantially inhibited the growth of Pestalotiopsis, even at low levels.
The next steps
Pestalotiopsis is recognized as a “weak” pathogen, and it may require certain conditions to become a problem. Volenberg has conducted some research with Norton in a greenhouse environment that suggests the symptoms of Pestalotiopsis are transient and may succumb to summer heat. In both Missouri and Virginia, the fungus seemed to require rain to get sporulation. Consequently, Pestalotiopsis may not be a problem in dry climates such as California—although that remains to be seen, particularly in areas with significant fog in the fall.
In early September, Morton found that Alternaria, another “ubiquitous” fungus, was highly associated with bunch stem necrosis and berry shrivel this season. Alternaria species also can be saprophytic or opportunistic pathogens similar to Pestalotiopsis. According to Morton and Volenberg, it appears that some grape cultivars are more susceptible to Pestalotiopsis, and their initial observations suggest the same may be true of Alternaria.
The association of Alternaria with clusters displaying symptoms of red rachises suggests that more research needs to be done that focuses on why these saprophytic and opportunistic pathogens are not being controlled with current pest-management programs. Morton notes that once specific fungi are identified as potential pathogenic agents in the field, the next step is to do controlled experiments to verify their pathogenicity.
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Based on observations made in Maryland in September 2017, Morton reports that Cabernet Sauvignon remains the variety with by far the most susceptibility to cluster stem wilt. The Maryland grower was not able to stop this from occurring again with applications of pre-bud break lime sulfur and a pre-bloom spray of Pristine. In view of this, Morton believes that more attention should be paid to the black-dot pycnidia present on the vine phloem/bark tissues. They are enclosed sacks of fruiting bodies that lie just beneath the epidermis and may be impervious to fungicides. She is hopeful that greenhouse tests both in her laboratory in Charlottesville and at Virginia Tech will help shed light on what turns “Pesta” and Alternaria from benign to pathogenic.
Volenberg’s projects involving Pestalotiopsis include:
1. Determining the environmental conditions that are conducive to Pestalotiopsis infection.
2. Identifying fungicides that provide control of Pestalotiopsis.
3. Identifying Pestalotiopsis species involved in grapevine infections.
Morton’s projects involving Pestalotiopsis include:
1. Evaluating new test results on buds and another year of red-stemmed cluster stem wilt.
2. Working with plant pathologists at Virginia Tech to study a potential link between Pestalotiopsis and bud necrosis.
3. Looking at the possibility of mite involvement with spreading the fungus.
References:
Guba, E.F., 1961. “Monograph of Pestalotia and Monochaetia.” Harvard University Press, Cambridge.
Steyaert, R.L., 1949. Contributions à l’étude monographique de Pestalotia de Not, et Monochaetia Sacc. (Truncatella gen. Nov. Et Pestalotiopsis gen nov.). Bulletin Jardin Botanique ?tat Bruxelles 19 :285-354.
Urbez-Torres, J.R.; F. Peduto, R.K. Striegler, K.E. Urrea-Romero, J.C. Rupe, R.D. Cartwright and W.D. Gubler, 2012. « Characterization of fungal pathogens associated with grapevine trunk diseases in Arkansas and Missouri.” Fungal Diversity. 52:169-189.
Volenberg, D.S. Vinews Viticulture Information News: Week of July 6, 2015; Week of May 30, 2016, Week of June 13, 2016; Week of June 2
7, 2016.
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