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Grapevine trunk diseases (GTDs) attack the vine's wood, devastating vineyards worldwide. Chile is the world's fourth-largest wine exporter, and Cabernet Sauvignon is one of the most economically important red wine varieties. Botryosphaeria dieback is an important GTD, and Diplodia seriata is one of the main pathogenic species. Biocontrol studies of these pathogens are commonly carried out at different incubation times but at a single temperature. This study aimed to evaluate the biocontrol effect of Chilean PGPB and grapevine endophytic bacteria against D. seriata at different temperatures. We analyzed the biocontrol effect of Pseudomonas sp. GcR15a, Pseudomonas sp. AMCR2b and Rhodococcus sp. PU4, with three D. seriata isolates (PUCV 2120, PUCV 2142 and PUCV 2183) at 8, 22 and 35 °C. Two dual-culture antagonism methods (agar plug diffusion and double plate) were used to evaluate the in vitro effect, and an in vivo test was performed with Cabernet Sauvignon cuttings. In vitro, the greatest inhibitions were obtained using the agar plug diffusion method and at a temperature of 8 °C, where Rhodococcus sp. PU4 obtains a 65% control (average) and Pseudomonas sp. GcR15a a 57% average. At 22 °C, only strains of Pseudomonas sp. show control. At 35 °C, one Pseudomonas strain shows the highest control (38%), on average, similar to tebuconazole (33%), and then Rhodococcus sp. (30%). In vivo, a biocontrol effect is observed against two D. seriata isolates, while the PUCV 2142 proves to be more resistant to control. The biocontrol ability at low temperatures is promising for effective control in the field, where infections occur primarily in winter.
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AIM: This study evaluates the in vitro efficacy of 8-hydroxyquinoline (8HQ) derivatives in controlling the phytopathogenic fungus Phaeomoniella chlamydospora. METHODS AND RESULTS: The in vitro tests assessed the susceptibility to the minimum inhibitory concentration (MIC), checkerboard assay, mycelial growth (MG) inhibition, and EC50 determination. Among the seven agricultural fungicides tested, tebuconazole (TEB) displayed the lowest MIC, 1.01 µg mL-1, followed by captan (CAP), thiophanate methyl (TM), and mancozeb with MICs of 4.06, 5.46, and 10.62 µg mL-1, respectively. The 8HQ derivatives used in this study were clioquinol and PH 151 (PH) with MICs of 1.09 and 2.02 µg mL-1, respectively. PH associated with TEB and CAP showed synergism and inhibited 95.8% of MG at the highest dose. TEB inhibited 100% of MG at the three highest doses, while associated with PH exhibited the lowest EC50 (0.863 + 0.0381 µg mL-1). CONCLUSIONS: We concluded that the 8HQ derivatives tested controlled effectively the P. chlamydospora in vitro. PH associated with CAP and TEB exhibited a synergistic effect. The association between PH and TM was considered indifferent. IMPACT STATEMENT: This study expands the list of active ingredients tested against P. chlamydospora, with the PH 151 and clioquinol derivatives being tested for the first time. The in vitro efficacy and synergistic action with other fungicides suggest a potential use as a grapevine wound protectant. This association makes it possible to reduce doses and increase the potency of both drugs, reducing the risk of resistance development and harm to humans and the environment.
Assuntos
Ascomicetos , Clioquinol , Fungicidas Industriais , Humanos , Fungicidas Industriais/farmacologia , Clioquinol/farmacologia , Oxiquinolina/farmacologiaRESUMO
Grapevine trunk diseases (GTDs) are one of the most important phytosanitary problems that affect grapevines (Vitis vinifera) worldwide. In Chile, Phaeomoniella chlamydospora is the major fungal trunk pathogen associated with GTDs. In the vineyards, the natural infections by P. chlamydospora are associated with air-borne conidia dispersed onto fresh pruning wounds from pycnidia. These pruning wounds are considered an important entrance for fungal trunk pathogens such as P. chlamydospora in the host in the field. However, the duration of the susceptibility of grapevine annual pruning wounds to P. chlamydospora is still unknown in Chile. Therefore, this study aimed to evaluate the period of susceptibility of pruning wounds of different ages to artificial infection of P. chlamydospora on grapevine cv. Cabernet Sauvignon, Central Chile. Artificial inoculations of a conidial suspension (105 conidia/mL) of P. chlamydospora were used to determine the susceptibility of pruning wounds of different ages, from 1, 15, 30, and 45 days after pruning. The experiments were conducted on lignified cuttings in a greenhouse, and on vine spurs in two vineyards (Buin and Nancagua, Central Chile) during two consecutive seasons. The results indicated that the pruning wounds of grapevine cv. Cabernet Sauvignon were very susceptible to infections by P. chlamydospora, with a percentage of pruning wounds infected from 97 to 71% for cuttings, and 96% to 60% for spurs, during the first 15 days after pruning. However, the susceptibility of pruning wounds of different ages in cuttings and spurs of grapevine, generally decreased as the time from pruning to inoculation increased. Moreover, the pruning wounds the pruning wounds remained susceptible to artificial inoculation by P. chlamydospora for up 45 days after pruning with percent of wounds infected from 8.0 to 12.2, and 8.3 to 18.8% on cuttings and spurs of grapevine, respectively. Finally, this study constitutes study constitutes the first research focalized on the susceptibility of pruning wounds of various ages of grapevine cv. Cabernet Sauvignon to artificial inoculations by P. chlamydospora in Central Chile.
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Grapevine trunk diseases threaten wine and table grape production worldwide, primarily by reducing yields and, in its advanced stages, causing plant death. Among those diseases, the complex etiology disease known as hoja de malvón (HDM) significantly concerns Argentinian and Uruguayan viticulture. At least four different fungi are associated with this disease, but their role and interactions with other wood microorganisms are understudied. In this sense, analyzing grapevine wood microbiome composition could help understand microbial interactions occurring in HDM onset. Hence, a metatranscriptomic study was performed for the microbiome characterization of mature field-grown Vitis vinifera cv. Malbec, leaf-symptomatic or leaf-asymptomatic. The microbiome was mainly represented by Dothideomycetes and Actinobacteria. In the plant with more marked symptoms, higher levels of the Basidiomycota Arambarria destruens and Phellinus laevigatus were detected. Despite this particular difference, discriminating symptomatic from asymptomatic plants based on the presence or abundance of HDM pathogens was not possible. Alpha diversity and rank-abundance curve analyses indicated that plants with foliar symptoms have lower microbial evenness than asymptomatic plants. The co-occurrence network modeled microbial interkingdom interactions. Molecular data generated in this study will help develop future targeted molecular quantification for specific taxa.
Assuntos
Ascomicetos , Microbiota , Vitis , Microbiota/genética , Doenças das Plantas/microbiologia , Vitis/microbiologia , Madeira/microbiologiaRESUMO
Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial ß-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and ßtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.
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Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.
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Phytotoxic metabolites produced in liquid culture by six species of Lasiodiplodia isolated in Brazil and causing Botryosphaeria dieback of grapevine were chemically identified. As ascertained by LC/MS, L. brasiliense, L. crassispora, L. jatrophicola, and L. pseudotheobromae produced jasmonic acid, and L. brasiliense synthesized, besides jasmonic acid, also (3R,4S)-4-hydroxymellein. L. euphorbicola and L. hormozganensis produced some low molecular weight lipophilic toxins. Specifically, L. euphorbicola produced (-)-mellein, (3R,4R)-(-)- and (3R,4S)-(-)-4-hydroxymellein, and tyrosol, and L. hormozganensis synthesized tyrosol and p-hydroxybenzoic acid. This is the first report on the production of the above cited metabolites from L. euphorbicola and L. hormozganensis. The phytotoxic activity of the metabolites produced is also discussed and related to the symptoms these pathogens cause in the grapevine host plants.