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.

First report of nut rot caused by Neofusicoccum parvum on hazelnut (Corylus avellana) in Italy

Hazelnut (Corylus avellana L.), which is native to Europe and Western Asia, is a widely distributed and economically important crop in Italy, cultivated on 82,104 ha and its production is 110,618 t (ISTAT 2021). A total of one hundred and eleven black rotted nuts (incidence: 41%) with sunken lesions from Lu and Cuccaro (45°00'21.8"N/8°28'59.6"E), north-western Italy, were collected during the ripening stage of hazelnuts during October-December 2020. Symptomatic half cut kernels were sterilized in 1% NaClO for 1 min, washed in sterile water twice, and dried on sterile filter paper. The fragments were placed onto potato dextrose agar (PDA) containing streptomycin. After 48 to 72 h of incubation at 25°C, fast-growing white colonies with abundant aerial mycelia were observed. On the opposite side of the plates, colonies were initially white, then turned to dark-grayish olive after one week of incubation. Dark colonies produced globose, hyaline, ellipsoidal, unicellular conidia ranging from 12.23-15 μm x 5-6.71 μm. Morphologically, the causal agent was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Zhang et al. 2021). The DNA from the isolates HMa-19-2 and Hwb-4b-2 was extracted with the E.Z.N.A. Fungal DNA mini kit (Omega Bio-tek) according to manufacturer instructions. Molecular identification was confirmed by sequencing of rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990) and translation elongation factor 1-alpha (tef-1α) gene by using the primers EF1-728F/EF1-986R (Carbone & Kohn, 1999). The sequences of both isolates were deposited in GenBank for ITS (accession numbers MZ848132 and MZ848133) and for tef-1α gene (accession numbers MZ913266 and MZ913267). The BLAST analysis showed 99% identity with ex-type strain of N. parvum (CMW9081) for ITS and tef-1α. Maximum likelihood method based on combined sequences of ITS and tef-1α genes was performed and the isolates of N. parvum clustered with ex-type strain of N. parvum (CMW9081; eXtra file). Pathogenicity of both isolates were tested on ripening hazelnuts (BBCH: 85) to evaluate Koch’s postulates. Three nuts per isolate, and per three replicates, were surface disinfected with 1% NaClO. A piece of shell (5 mm diameter) from nuts was removed with a sterile cork borer, then nuts were inoculated with PDA mycelium plugs of the same diameter cut from 7 days old PDA colony (Seddaiu et al. 2021). The control nuts were treated with sterilized PDA plugs. All inoculated nuts produced black lesions with softening pulp (eXtra file). Additionally, abundant white-gray mycelium developed on the inoculation sites. Control nuts showed no symptoms. Neofusicoccum parvuwas recently reported in Italy on chestnut (Seddaiu et al. 2021) and blueberry (Guarnaccia et al. 2021). The fungal pathogen was also reported as agent of grey necrosis of hazelnut in Chile (Duran et al. 2020). However, this is the first report of N. parvum on hazelnut nut in Italy. The findings suggest that N. parvum could severely affect hazelnut production in Italy. Accurate identification of the pathogen will support the growers to manage the disease.

Trichoderma spp. from Pine Bark and Pine Bark Extracts: Potent Biocontrol Agents against Botryosphaeriaceae

Pinus sylvestris bark represents a rich source of active compounds with antifungal, antibacterial, and antioxidant properties. The current study aimed to evaluate the antifungal potential of P. sylvestris bark against Botryosphaeria dothidea, Dothiorella sarmentorum, and Neofusicoccum parvum (Botryosphaeriaceae) through its chemical (water extracts) and biological (Trichoderma spp. isolated from the bark) components. The water bark extracts were prepared at two temperatures (80 and 120 °C) and pH regimes (7 and 9). The presence of bark extracts (30%) caused inhibition of mycelial growth of B. dothidea and D. sarmentorum for 39 to 44% and 53 to 60%, respectively. Moreover, we studied the antagonistic effect of three Trichoderma isolates originating from the pine bark. Trichoderma spp. reduced growth of B. dothidea by 67%–85%, D. sarmentorum by 63%–75% and N. parvum by 55%–62%. Microscopic examination confirmed typical mycoparasitism manifestations (coiling, parallel growth, hook-like structures). The isolates produced cellulase, β-glucosidase and N-acetyl-β-glucosaminidase. The volatile blend detected the emission of several volatile compounds with antimicrobial activity, including nonanoic acid, cubenene, cis-α-bergamotene, hexanedioic acid, and verticillol. The present study confirmed in vitro potential of P. sylvestris bark extracts and Trichoderma spp. against the Botryosphaeriaceae. The study is an important step towards the use of environmentally friendly methods of Botryosphaeriaceae disease control.

Insights of the Neofusicoccum parvum–Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch’s postulates—followed by a molecular approach—revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue–N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction.

Hunting the plant surrender signal activating apoplexy in grapevines after Neofusicoccum parvum infection

Apoplectic breakdown from Grapevines Trunk Diseases (GTDs) has become a serious challenge to viticulture in consequence to drought stress. We hypothesise that fungal aggressiveness is controlled by a chemical communication between host and colonising fungus. We introduce the new concept of a "plant surrender signal" accumulating in host plants under stress and triggering aggressive behaviour of the strain Neofusicoccum parvum (Bt-67) causing Botryosphaeriaceae-related dieback in grapevines. Using a cell-based experimental system (Vitis cells) and bioactivity-guided fractionation, we identify trans-ferulic acid, a monolignol precursor, as "surrender signal". We show that this signal specifically activates secretion of the fungal phytotoxin Fusicoccin A. We show further that this phytotoxin, mediated by 14-3-3 proteins, activates programmed cell death in Vitis cells. We arrive at a model pinpointing the chemical communication driving apoplexy in Botryosphaeriaceae-Vitis interaction and define the channelling of phenylpropanoid pathway from the lignin precursor, trans-ferulic acid to the phytoalexin trans-resveratrol as target for future therapy.

Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum

Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.