Biological and chemical pruning wound protectants reduce infection of grapevine trunk disease pathogens

Grapevine trunk diseases (GTDs) are currently considered some of the most important challenges for viticulture, curtailing vineyard longevity and productivity in nearly every raisin, table and wine grape production region in California and worldwide. Pruning wounds provide the main entry point for fungal pathogens responsible for these diseases; pathogens enter the wounds following precipitation events. The aim of this study was to evaluate the efficacy of selected chemical and experimental biological fungicides for protection of pruning wounds against two of the most common and virulent fungal pathogens causing GTDs: Eutypa lata and Neofusicoccum parvum. This study was conducted on sauvignon blanc at the UC Davis Department of Plant Pathology Field Station. Results showed that several chemical and biological fungicides, notably the chemical fungicide Luna Sensation, the biofungicide Vintec and a combination of the biofungicides Bio-Tam and CrabLife Powder, provided significant protection against at least one of the two canker pathogens used in this study. However, the majority of products tested did not provide simultaneous control of both E. lata and N. parvum pathogens, highlighting the continuing challenge of controlling GTDs.

Evaluation of Biological and Chemical Pruning Wound Protectants to Control Grapevine Trunk Diseases Pathogens Eutypa lata and Neofusicoccum parvum

Grapevine trunk diseases, caused by many different fungal pathogens, are one of the most economically important diseases affecting the grapevine industry worldwide. Pruning wounds are the main point of entry for these fungal pathogens and thus, disease control is focused on preventative pruning wound protection by chemical products and/or biological control agents (BCAs). In this study we evaluated a broad variety of already registered or at the experimental stage of chemical and BCAs in greenhouse and in field trials for the protection of table- and wine-grape vines against infection of Eutypa lata and Neofusicoccum parvum, major pathogens responsible for Eutypa and Botryosphaeria dieback, respectively. Our study showed that Trichoderma asperellum and Trichoderma gamsii consistently provided pruning wound protection in greenhouse and field trials, with a mean percent disease control (MPDC) of 88% and 100% for E. lata and N. parvum, respectively, when compared to the water treated-inoculated positive control (P<0.05). The chemical protectants, thiophanate-methyl + myclobutanil and fluopyram and trifloxystrobin were also able to effectively protect wounds with a MPDC of up to 86% when compared to the water treated-inoculated positive control (P<0.05). When biological treatments were evaluated for recovery from treated canes at the end of the growing season, Trichoderma-based treatments had a rate of recovery between 0 and 100%, Aureobasidium-based treatments had a recovery rate between 25 and 100%, and Bacillus-based treatments had a recovery rate between 0 and 25%.

Pathogenicity testing of fungal isolates associated with olive trunk diseases in South Africa

A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified several fungi associated with olive trunk disease symptoms, including species of Basidiomycota, Botryosphaeriaceae, Coniochaetaceae, Calosphaeriaceae, Diaporthaceae, Diatrypaceae, Phaeomoniellaceae, Phaeosphaeriaceae, Symbiotaphrinaceae, Togniniaceae and Valsaceae. Many of the species recovered had not yet been reported from olive trees and therefore the aim of this study was to determine their pathogenicity towards this host. Pathogenicity tests were first conducted on detached shoots to select virulent isolates which were then used in field trials. During field trials, 2-year-old olive branches of 15-year-old trees were inoculated by inserting colonised agar plugs into artificially wounded tissue. Measurements were made of the internal lesions after 8 months. In total, 58 isolates were selected for the field trials. Species that formed lesions significantly larger than the control could be considered as olive trunk pathogens. These include Biscogniauxia rosacearum, Celerioriella umnquma, Coniochaeta velutina, Coniothyrium ferrarisianum, isolates of the Cytospora pruinosa complex, Didymocyrtis banksiae, Diaporthe foeniculina, Eutypa lata, Fomitiporella viticola, Neofusicoccum stellenboschiana, Nm. vitifusiforme, Neophaeomoniella niveniae, Phaeoacremonium africanum, Pm. minimum, Pm. oleae, Pm. parasiticum, Pm. prunicola, Pm. scolyti, Pm. spadicum, Pleurostoma richardsiae, Pseudophaeomoniella globosa, Punctularia atropurpurascens, Vredendaliella oleae, an undescribed Cytospora sp., Geosmithia sp., two undescribed Neofusicoccum spp. and four Xenocylindrosporium spp. Pseudophaeomoniella globosa can be regarded as one of the main olive trunk pathogens in South Africa, due to its high incidence from olive trunk disease symptoms in established orchards and due to its high virulence in pathogenicity trials.

First report of Eutypa lata causing cankers and dieback in sweet cherry in Chile

Sweet cherry (Prunus avium) is one the most important fruit crops in Chile. Its production has significantly grown in recent years, reaching 228,448 tons exported in 2019/2020, to 47 countries. One of the main threats for this expanding crop are fungal pathogens, especially those that cause wood diseases. Cherry orchards (n=35) located in the central area of Chile, from Curicó (34°58'58''S 71°14.366'W) to Angol (37°47'42.7''S 72°42.982'W), were surveyed during 2020. Wood samples were collected (n= 72) from living branches and trunks showing dieback, cankers and dark necrosis, mostly wedge shaped. Small wood sections (0.5-cm) were cut off from the margin of the necrosis and surface disinfected using 0.5% v/v sodium hypochlorite. Sections were plated on a quarter-strength potato dextrose agar amended with 1mg/L tetracycline (PDA-tet). Plates were incubated at 25°C until mycelial development and subsequently the isolates were purified transferring excised fungal tips to PDA. Colonies (n=21) developed white cottony mycelia, which turned slightly greyish and flatter after 10-days at 25°C. Isolates developed black pycnidia which released beige conidial matrixes after subsequent 15-days at 25 +/-2°C and 12-h photoperiod. Conidia were hyaline, curved and filiform, measuring 19.8-(27.9)-36.7 μm length (lineal) x 1.2-(1.7)-1.9 μm width (n=70), according to Eutypa lata (Rappaz, 1984). DNA was extracted from mycelia of the representative isolates HMCe30a, HMCe41a, HMCe109c and HMCe110a. The partial β-tubulin gene was amplified using bt2A/bt2B primers (Glass & Donaldson 1995) and the internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al. 1990). Sequences were BLAST analyzed, finding that ITS shared 99% and βTUB 100% identity with E. lata strain CBS 208.87 (Rolshausen et al. 2006). Sequences were accessioned to GenBank (MW363035, MW363034, MW363033 and MW363032 [ITS], and MW366820, MW366819, MW366818and MW366817 [βTUB]). The isolates were inoculated on sweet cherry healthy plants cv. Kordia, produced by rooting scions in tap water amended with 500 ppm of indole-butyric acid, for 30 days. An injury was made in the upper third of the shoot using a sterile 0.5-cm diameter corkborer. Mycelial plugs were placed on the injuries and covered with plastic film, using sterile agar for controls (n=25). Plants were incubated in aerated tap water for 60 days at 23 +/-3 °C. After incubation, plants were cut exposing dark-brown necrotic lesions, while control plants remained asymptomatic. Moreover, 2-year old potted plants cv. Lapins were inoculated (n=3 per isolate) with mycelial plugs, on fresh cuts of their main lateral branches, in January 20th, and remained under partial shade for 72-days. After incubation, bark was removed from inoculated branches and the necrotic lesions length was measured. HMCe109c was the most virulent isolate (3.6 cm), followed by HMCe30a (2.1 cm), HMCe41a (1.9 cm) and HMCe110a (1.1 cm), while symptoms were not reproduced in controls. Fulfilling Koch’s postulates, fungi were reisolated from all inoculated plants in both pathogenicity tests and no fungus was recovered from controls. To our knowledge this is the first report of Eutypa lata causing wood decay in sweet cherry in Chile. The pathogen was recently reported causing dieback of grapevines in Chile (Lolas et al. 2020). These are significant findings due to the frequent proximity of sweet cherry orchards and vineyards, which facilitates cross infections.

Fungal Pathogens Associated With Canker Diseases of Almond in California

Almond canker diseases are destructive and can reduce the yield as well as the lifespan of almond orchards. These diseases may affect the trunk and branches of both young and mature trees and can result in tree death soon after orchard establishment in severe cases. Between 2015 and 2018, 70 almond orchards were visited throughout the Central Valley of California upon requests from farm advisors for canker disease diagnosis. Two major canker diseases were identified, including Botryosphaeriaceae cankers and Ceratocystis canker. In addition, five less prevalent canker diseases were identified, including Cytospora, Eutypa, Diaporthe, Collophorina, and Pallidophorina canker. Seventy-four fungal isolates were selected for multilocus phylogenetic analyses of internal transcribed spacer region ITS1-5.8S-ITS2 and part of the translation elongation factor 1-α, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase gene sequences; 27 species were identified, including 12 Botryosphaeriaceae species, Ceratocystis destructans, five Cytospora species, Collophorina hispanica, four Diaporthe species, two Diatrype species, Eutypa lata, and Pallidophorina paarla. The most frequently isolated species were Ceratocystis destructans, Neoscytalidium dimidiatum, and Cytospora californica. Pathogenicity experiments on almond cultivar Nonpareil revealed that Neofusicoccum parvum, Neofusicoccum arbuti, and Neofusicoccum mediterraneum were the most virulent. Botryosphaeriaceae cankers were predominantly found in young orchards and symptoms were most prevalent on the trunks of trees. Ceratocystis canker was most commonly found in mature orchards and associated with symptoms found on trunks or large scaffold branches. This study provides a thorough examination of the diversity and pathogenicity of fungal pathogens associated with branch and trunk cankers of almond in California.

The influence of water deficit stress on the grapevine trunk disease pathogens Eutypa lata and Diplodia seriata

The increasing prevalence of the grapevine trunk diseases Eutypa and Botryopshaeria dieback has been attributed, in part, to abiotic stresses imposed on vineyards as production intensifies worldwide. The aim of this study was to evaluate the influence of water deficit irrigation practices on the infection of pruning wounds by Eutypa lata and Diplodia seriata, and the subsequent rate of colonisation. Two vineyard trials were conducted over 2 years in South Australia, one in the Riverland using ‘Cabernet Sauvignon’ with four irrigation treatments (100, 50, 25 and 12.5% of the standard irrigation program) and another in the Barossa Valley using ‘Shiraz’ on six rootstocks and own roots, either irrigated or not irrigated. According to leaf water potential assessments, vines with reduced irrigation were generally in water deficit, and therefore subjected to stress. On the whole, incidence of wound infection and distance of colonisation were similar among irrigation treatments for both pathogens, except in the Riverland, where E. lata colonized canes to a greater extent in well-watered vines than those in water deficit. Only vines on rootstock ‘Ramsey’ in the Barossa Valley had greater extent of colonisation by E. lata in the non-irrigated vines. There was no correlation between internal staining and colonisation, with both pathogens recovered up to nearly 20 cm ahead of the staining. Water deficit did not increase the susceptibility of grapevine pruning wounds to infection, nor colonisation of the subtending tissue by E. lata and D. seriata. In fact, there was evidence of decreased susceptibility to colonisation by E. lata in vines subjected to severe water deficit.

Hunting modulators of plant defence: the grapevine trunk disease fungus Eutypa lata secretes an amplifier for plant basal immunity

Grapevine trunk diseases (GTDs) are progressively affecting vineyard longevity and productivity worldwide. To be able to understand and combat these diseases, we need a different concept of the signals exchanged between the grapevine and fungi than the well-studied pathogen-associated molecular pattern and effector concepts. We screened extracts from fungi associated with GTDs for their association with basal defence responses in suspension cells of grapevine. By activity-guided fractionation of the two selected extracts, O-methylmellein was identified as a candidate modulator of grapevine immunity. O-Methylmellein could not induce immune responses by itself (i.e. does not act as an elicitor), but could amplify some of the defence responses triggered by the bacterial elicitor flg22, such as the induction level of defence genes and actin remodelling. These findings show that Eutypa lata, exemplarily selected as an endophytic fungus linked with GTDs, can secrete compounds that act as amplifiers of basal immunity. Thus, in addition to elicitors that can trigger basal immunity, and effectors that down-modulate antibacterial basal immunity, once it had been activated, E. lata seems to secrete a third type of chemical signal that amplifies basal immunity and may play a role in the context of consortia of mutually competing microorganisms.