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Hazelnut is among the most important nut crops in Chile, currently covering 46,000 ha. In 2023, the country exported 30,000-ton. In recent years the incidence of plants with internal discoloration, cankers and dieback has been increasing. In some cases, the trees died and had to be removed and, after a year, purple resupinate fruiting bodies were observed growing from the stumps. To determine the etiology of the symptoms and signs, wood samples (n=318) were collected since 2020, from 38 symptomatic orchards from Maule to La Araucanía Regions, primarily from the cvs. Tonda di Giffoni and Lewis. Wood sections 0.5 cm diameter were cut from the symptomatic tissues, disinfected using a sodium hypochlorite (10%) solution, and plated on a quarter-strength acidified potato dextrose agar (aPDA1/4). The plates were incubated and purified on PDA. Subsequently, isolates were identified by morphological and molecular means. Almost half of the isolates (47%) were preliminarily identified as basidiomycetes, based on mycelial features such as the presence of clamp connections, with 45% of them exhibiting abundant whitish cottony fast-growth mycelia, resembling Chondrostereum purpureum (Grinbergs et al., 2020). DNA was extracted and the 500-bp fragment, located between 5S and 18S ribosomal regions, was amplified using APN1 specific primers (Becker et al. 1999), identifying the isolates as C. purpureum. In addition, 5.8S gene of RGM1 (35°13'40.9"S 71°25'14.1"W), RGM2 (36°31'27.95"S 71°46'58.31"W), RGM3 (37°10'54.8"S 72°03'39.6"W), RGM4 (35°19'25.2"S 71°19'54.7"W) and RGM5 (36°35'30.8"S 72°05'18.8"W) isolates, representing different locations within the hazelnut growing area, was amplified using ITS1/ITS4 primers (White et al., 1990). The PCR product was sequenced, and the analysis showed 100% homology among isolates (Genebank codes: PP839283, PP839284, PP839285, PP839286 and PP839287, respectively). To determine the pathogenicity of the isolates, 30-cm healthy cuttings cv. Lewis were inoculated with mycelial plugs, while control shoots were inoculated with sterile agar plugs. Cuttings were vertically arranged in pots with 3-cm water and incubated for 60-d at 22°C. In addition, fresh cuts of 3-y potted plants cv. Lewis were inoculated with mycelial plugs and incubated for 137-d in a shadehouse. After incubation, bark was removed from inoculated cuttings and the length of necrotic lesions was measured. Although discoloration was reproduced by all the isolates in both pathogenicity tests, RGM1 isolate was the most aggressive, causing the complete discoloration of the cuttings and the death of the inoculated plants. To our knowledge this is the first report of C. purpureum causing wood disease in hazelnut. These findings are significant because the disease may not only reduce orchard longevity but also decrease fruit yield and quality, as observed in other fruit crops (Grinbergs et al., 2021).
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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
Botryosphaeria dieback (BD) occurs in young and old plants. In the field, the prevalence and severity of the disease increase proportionally with the age of vineyards. Among the pathogens that cause BD, Diplodia seriata is the most prevalent species in Chile and other countries with a Mediterranean climate. To date, no information is available on the susceptibility of adult wood to infection by this pathogen since most of the pathogenicity tests have been carried out on 1- or 2-year-old shoots or detached canes. Therefore, a pathogenicity test was carried out on plants under field conditions, with inoculations in 1-year-old shoots and 2- and 10-year-old wood in grapevine cv. Cabernet Sauvignon. A pathogenicity test was carried out with two isolates of D. seriata. The results for the plants show that D. seriata was significantly more aggressive on the 10-year-old than on the one- or two-year-old tissue, where the lesions were 4.3 and 2.3 cm on average, respectively. These results were compared with the lesions obtained from two-year-old canes after the isolates were activated in grape berries. Also, the Chilean isolates of D. seriata were compared phylogenetically with those from other countries, and no major differences were found between them. Our results are consistent with the damage observed in the field, contributing to the knowledge of the epidemiology of this disease in Mediterranean climates. In the future, the effect observed in cv. Cabernet Sauvignon with D. seriata on virulence at different tissue ages should be tested for other BD-causing agents and wine varieties.
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Peach (Prunus persica) is an important stone fruit crop in Chile, with 7,665 h in 2022. Trunk diseases symptoms, including shoot dieback, longitudinal cankers and internal dark-brown to purple discolorations in cross sections were observed in a commercial orchard, in March of 2021. In severe cases, mostly in old trees, periderm sections were detached from the cankers, showing circinate groups of black long necked perithecia. To isolate the causal agent, wood samples were collected from March 2021 to October 2022, from symptomatic trees (n=23) of commercial orchards (n=12) (34°12'36.47"S 70°46'3.43"O to 34°34'26.48"S 70°58'17.97"O), located in O'Higgins Region, in the center of Chile. Isolations were performed cutting wood sections (0.5 cm) from the necrosis progress area, disinfecting them in sodium hypochlorite (10%), plating on a quarter-strength potato dextrose agar amended with 1 mg/L tetracycline (aPDA-tet) and incubating at 25°C, until mycelial development. Cultures were purified on PDA and identified by morphological means. Colonies on PDA were dark-pink and purple to orange-red, with regular margins, usually white, and produced abundant hyaline ellipsoidal to allantoid conidia (3.8-5.7 × 1.3 µm). In some cases, perithecia developed on disinfected wood on culture media, showing clavate unitunicate asci with hyaline allantoid ascospores 4.5-6.2 × 0.7 µm. The morphological characteristics of anamorph and teleomorph structures of field and culture isolates accorded to those described for Calosphaeria pulchella (1,2). DNA from representative isolates was extracted and the ITS region was amplified by PCR using ITS1/ITS4 primers (3), sequenced and BLAST analyzed. BLAST results revealed that ITS sequences identity of the representative isolates HMDu263 and HMDu271, shared 99 and 100% similarity, respectively, when compared to isolate CBS115999 (EU367451) (1,2,4). Sequences were accessioned to GenBank (OP216663 and OP216664 [ITS]). To determine the pathogenicity of C. pulchella, 17 representative isolates were inoculated on peach canes (25 cm) (n=7 per isolate), that were previously rooted on tap water amended with 500 ppm of indole-butyric acid, for 30 d. Mycelial plugs (0.5 cm) from actively growing colonies on PDA were placed on circular injuries made in the upper third of the shoots using a sterile corkborer and covered with plastic film. Sterile agar was used for controls. After 60 d of incubation in aerated tap water, at 23 +/-3 °C, bark was removed, and the necrosis length was measured and compared. Mean length values of lesions went from 9.5 to 27 cm. The most virulent isolates (n=7) were inoculated on fresh cuts of main shoots of nursery plants (n=3 per isolate) cv. Royal Glory, with 200 µL conidial suspensions (1x105 conidia/mL), on March 18th, 2022. Plants were incubated at shadehouse for 102 d and after the incubation period, shoots were cut (30 cm), bark was removed, and discoloration length was measured. All the isolates were pathogenic, with differences among their virulence (ANOVA, LSD, P < 0.05) from 5.2 cm (HMDu246) to 24.3 cm (HMDu266). Fungus was successfully reisolated from symptomatic canes (100%) and trees (98.7%), but not from the controls in both pathogenicity tests, fulfilling Koch's postulates. Calosphaeria pulchella was recently reported causing trunk disease in sweet cherry in Chile (4) and these results contribute to the knowledge of trunk diseases of fruit crops in Chile and to the understanding of the pathogen worldwide.
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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.
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Ascomicetos , Microbiota , Vitis , Microbiota/genética , Doenças das Plantas/microbiologia , Vitis/microbiologia , Madeira/microbiologiaRESUMO
Silverleaf is caused by the fungus Chondrostereum purpureum, which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Because plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased, and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum, and defense genes expression. Water potential, stomatal conductance, chlorophyll content, and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by denaturing gradient gel electrophoresis, and community richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum-virulent strains in dual-culture assays. Defense genes expression was measured by quantitative PCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal community richness was similar in healthy and reverted plants and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum. Furthermore, nonexpressor of pathogenesis-related gene 1 expression was upregulated in reverted plants, whereas phenylalanine ammonia lyase and polygalacturonase-inhibiting protein genes showed higher values in diseased plants. Overall, physiological, molecular, and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum.
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Malus , Doenças das PlantasRESUMO
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.
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Ascomicetos/metabolismo , Micotoxinas/metabolismo , Vitis/microbiologia , Ascomicetos/química , Ascomicetos/patogenicidade , Brasil , Cromatografia Líquida de Alta Pressão , Ciclopentanos/metabolismo , Isocumarinas/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Micotoxinas/química , Ocratoxinas/metabolismo , Oxilipinas/metabolismo , Parabenos/metabolismo , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Doenças das Plantas/microbiologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Perennial cankers and consequent grapevine dieback are a major problem in vineyards of Sonora and Baja California, the most important grape-production areas of Mexico. In order to identify the canker-causing agents, symptomatic arms, cordons, and trunks were collected from 13 and 6 vineyards in Sonora and Baja California, respectively. Two Botryosphaeriaceae spp., Lasiodiplodia theobromae and Diplodia seriata, were isolated frequently from infected wood and identified based on morphological and cultural characters as well as analyses of nucleotide sequences of three genes, the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the ß-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Although both L. theobromae and D. seriata were isolated from grapevine cankers in Baja California, only L. theobromae was found in vines in the Sonora region. Pathogenicity of both species was verified by inoculation of rooted cuttings and green shoots of Thompson Seedless and Chardonnay cultivars. Isolates of L. theobromae were more virulent, based on the extent of spread in the secondary wood and green tissue, than those of D. seriata. These findings confirm L. theobromae and D. seriata as the causal agents of dieback and canker formation of grapevines in northern Mexico.