RESUMO
Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95 M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate.
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AIM: The aim was to characterize cold-adapted bacteria by testing their PGP features and antagonistic activity against Macrophomina phaseolina, both in vitro and coating soybean seeds (Glycine max [L.] Merr.). METHODS AND RESULTS: Burkholderia gladioli MB39, Serratia proteamaculans 136 and Serratia proteamaculans 137 were evaluated. In vitro tests showed that S. proteamaculans 136 and 137 produce siderophore and indole-acetic acid (IAA), solubilize phosphate and fix nitrogen. Additionally, B. gladioli MB39 and S. proteamaculans 137 showed hydrolase activity and potent antifungal effects. The biocontrol efficacy over soybean seeds was evaluated using in vitro and greenhouse methods by immersing seeds into each bacterial suspension. As a result, S. proteamaculans 136 has improved the performance in all the seed germination evaluated parameters. In addition, S. proteamaculans 137 and B. gladioli MB39 strongly inhibited M. phaseolina, reducing the infection index values to 10% and 0%, respectively. CONCLUSION: Serratia proteamaculans 136, 137 and Burkholderia gladioli MB39 showed plant growth promotion features and inhibition of Macrophomina phaseolina infection by producing different antifungal compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results reinforce the application of cold-adapted Serratia proteamaculans and Burkholderia gladioli bacterial strains as candidates for developing microbial formulation to promote plant growth and guarantee antifungal protection in soybean crops.
Assuntos
Glycine max , Doenças das Plantas , Glycine max/microbiologia , Doenças das Plantas/microbiologia , Sideróforos , Antifúngicos/farmacologia , Serratia , Sementes , Nitrogênio , Fosfatos , Acetatos , HidrolasesRESUMO
Stevia (Stevia rebaudiana [Bertoni] Bertoni) is a perennial plant originating in Paraguay. Stevia is primarily cultivated for the production of non-caloric sweeteners. In December 2018, wilted stevia cv. 'PC4' were recovered from two separate fields of 0.3 ha (24.66 S 56.46 W) and 0.5 ha (24.69 S 56.44 W), both with 3 years history of stevia production in San Estanislao County, San Pedro, Paraguay. The wilted plants were randomly distributed in beds covered with plastic mulch and a 30% disease incidence was recorded. Dark brown septate hyphae and microsclerotia were observed on stem bases and black necrotic roots of the wilted plants. Root and crown regions were washed, cut into 0.5 to 1.0 cm pieces, and then surface-disinfested with 0.6% NaOCl before placing them in Petri dishes containing acidified potato-dextrose-agar. Plates were incubated for one week at 25 ± 5°C under fluorescent light with a 12 h photoperiod yielding five isolates SP1PY, SP2PY, SP3PY, SP4PY and SP5PY with gray-black colonies without conidia but showing numerous microsclerotia. Twenty microsclerotia from pure cultures of five isolates were measured, with mean width 38.8 ± 4.7 µm and length 68.8 ± 15.5 µm. Fungal DNA was extracted from mycelia of five isolates for PCR amplification of the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) using ITS4/ITS5 and EF1-728F/EF-2 primers (Machado et al. 2019). The resultant amplicons were sequenced at Eton Bioscience (Research Triangle Park, NC) and deposited in the NCBI GenBank database (ITS: MT645815, OM956150, OM956151, OM956152, OM956153; and TEF1-α: MT659121, OM959505, OM959506, OM959507, OM959508). Sequences were aligned with several isolates of Macrophomina spp. previously reported (Huda-Shakirah et al. 2019; Machado et al. 2019; Santos et al. 2020; Poudel et al. 2021) using ClustalW. Alignments (ITS and TEF-1α) were concatenated to generate a maximum likelihood tree using MEGA7. The novel isolates grouped into the M. euphorbiicola clade with 95% of bootstrap support. Stevia plants cv. 'Katupyry' were grown in 10 cm-diameter nursery bags containing autoclaved sandy soil and kept under greenhouse conditions (28 ± 5°C; 16 h photoperiod). Fifteen plants per isolate (n=75) were inoculated by adding 20 g of rice infested with M. euphorbiicola to each plant. Infested grains were distributed around the crown of the plant at a depth of 0.5 cm; non-infested rice was added to four control plants. Lower-stem lesions and microsclerotia of M. euphorbiicola developed on all inoculated plants. No lesions or microsclerotia were observed on control plants. The M. euphoribiicola fungus was re-isolated from inoculated stevia plants but not from the non-infested rice treated plants. Koch's postulates were repeated twice with similar results. Previously, M. phaseolina was reported causing charcoal rot on stevia in Egypt (Hilal and Baiuomy 2000), and in North Carolina, USA (Koehler and Shew 2017). However, Paraguayan isolates grouped with isolates of M. euphorbiicola based on the combined sequences of the ITS and TEF-1α regions. Machado et al. (2019) reported M. euphorbiicola causing charcoal rot on castor bean (Ricinus communis) and bellyache bush (Jatropha gossypifolia) in Brazil, which borders northeast Paraguay, a major stevia production area. This pathogen has a significant impact on stevia production during hot, dry weather by reducing the number of harvestable plants and increasing replanting costs in perennial production systems.
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The fungi Macrophomina phaseolina is the charcoal rot causal agent, one of the most important cowpea crop disease in semiarid regions can causes 100% yield losses. The search for resistant genotypes requires efficient phenotyping. In addition, there is the problem of great variation in aggressiveness between isolates. This study aimed to 1) test three methods of inoculation in semiarid conditions, and 2) to evaluate the aggressiveness of isolates of M. phaseolina. In the first experiment carried out in greenhouse, the inoculations methods were evaluated, using two cowpea lines, three inoculation methods and three pathogen isolates. On the second experiment, fifteen M. phaseolina isolates were inoculated in one cultivar to evaluate their aggressiveness. By assessing the length of the lesions and the severity of the disease using an index, we identified the toothpick inoculation method as the most efficient. Toothpick method allowed to discriminate the genotypes and the aggressiveness of the pathogen.
O fungo Macrophomina phaseolina é o agente etiológico da podridão cinzenta do caule, uma das mais importantes doenças na cultura do feijão caupi em regiões semiáridas, podendo ocasionar perdas de 100%. A busca por genótipos resistentes exige uma fenotipagem eficiente. Além disso tem o problema da grande variação na agressividade entre isolados. Este trabalho teve como objetivos 1) testar três metodologias de inoculação em condições semiáridas, e 2) avaliar a agressividade de isolados de M. phaseolina. No primeiro experimento, conduzido em ambiente protegido, avaliou-se metodologias de inoculação em duas linhagens de caupi, por três métodos de inoculação e três isolados do patógeno. No segundo experimento, 15 isolados de M. phaseolina foram inoculados em uma cultivar de caupi para avaliar a agressividade do patógeno. Pela avaliação do comprimento das lesões e da severidade da doença por meio de um índice, identificamos o método de inoculação por palito de dente mais eficiente pois permitiu discriminar os genótipos estudados e a agressividade do patógeno.
Assuntos
Vigna , Fungos/genética , Fungos/patogenicidadeRESUMO
Charcoal rot is an emerging disease for peanut crops caused by the fungus Macrophomina phaseolina. In Mexico, peanut crop represents an important productive activity for various rural areas; however, charcoal rot affects producers economically. The objectives of this research were: (a) to identify and morphologically characterize the strain "PUE 4.0" associated with charcoal rot of peanut crops from Buenavista de Benito Juárez, belonging to the municipality of Chietla in Puebla, Mexico; (b) determine the in vitro and in vivo antagonist activity of five Trichoderma species on M. phaseolina, and (c) determine the effect of the incidence of the disease on peanut production in the field. Vegetable tissue samples were collected from peanut crops in Puebla, Mexico with the presence of symptoms of charcoal rot at the stem and root level. The "PUE 4.0" strain presented 100% identity with M. phaseolina, the cause of charcoal rot in peanut crops from Buenavista de Benito Juárez. T. koningiopsis (T-K11) showed the highest development rate, the best growth speed, and the highest percentage of radial growth inhibition (PIRG) over M. phaseolina (71.11%) under in vitro conditions, in addition, T. koningiopsis (T-K11) showed higher production (1.60 ± 0.01 t/ha-1) and lower incidence of charcoal rot under field conditions. The lowest production with the highest incidence of the disease occurred in plants inoculated only with M. phaseolina (0.67 ± 0.01 t/ha-1) where elongated reddish-brown lesions were observed that covered 40% of the total surface of the main root.
RESUMO
Peanut (Arachis hypogaea L.) is the third most important oilseed crop in the world. The cultivated area in Mexico is currently 52,046 ha with a production of 91,109 ton in 2018 (FAO, 2020). Puebla state ranks third in the national production with 9,313 ton (SIAP, 2020). In September 2019, typical symptoms of charcoal rot (Macrophomina phaseolina (Tassi) Goid.) were observed in about 50% of cultivar Virginia Champs peanuts, and it affecting 1.5 ha located in Chietla (18° 27' 39" N; 98° 37' 11" W), Puebla, Mexico. Diseased plants showed brown discoloration in stem and root rot, with chlorotic foliage, dark microsclerotia were observed on the stem and premature dying. To isolate the causal agent of these symptoms, 20 infected plants were recovered and processed in the laboratory. Ten pieces of stem and root tissue were selected from each plant, cut into small pieces 5-mm in length, superficially disinfested with 1% sodium hypochlorite for 3 min, followed by three rinses with sterile distilled water. Later, dried on sterile paper and placed on Petri plates containing potato dextrose agar (PDA) medium, which were kept at 28°C for 7 days (12 h light and 12 h dark). Four colonies were purified via hyphal tip culture, fungus was consistently isolated from the analyzed tissues; additional microcultures were prepared to observe phenotypic characteristics. Colonies showed dense growth, with a gray initial mycelium, becoming black after 7 days. Microesclerotia with spherical to oblong in shape were observed after 5 days on PDA, with a black coloration, measuring an average of 74 µm width × 110 µm length (n=40). Phylogenetic analysis was conducted by amplification and sequencing of the internal transcribed spacer (ITS) region with the ITS5 and ITS4 primers (White et al. 1990). The obtained sequences were deposited in GenBank database under accession numbers: MW585378, MW585379, MW585380, and MW585381 containing approximately 601 bp of the ITS1-5.8S-ITS2 region (complete sequence); they were 99% identical with the reference sequence of Macrophomina phaseolina (GenBank accession KF951698) isolated in Phaseolus vulgaris from Mexico. Based on the symptoms in the field, colony morphology, color, and shape of the microsclerotia, and molecular identification, the fungus was identified as M. phaseolina (Tassi) Goid. The pathogenicity test was performed on peanut plants cultivar Virginia Champs grown on plastic pots with an autoclaved peat/soil mixture under greenhouse conditions (70% relative humidity and 28°C). Fifty two-month-old peanut plants were inoculated using the toothpick method. The toothpicks were previously sterilized and then placed in Petri plates with each of the four colonies of M. phaseolina until colonization. Small wounds were made with those toothpicks in the roots, and a sterile toothpick was used in the control plants, the assays were performed twice. After three weeks, the inoculated plants exhibited symptoms of wilting chlorosis on the leaves and brown to dark brown discoloration of the vascular ring, while control plants remained healthy. M. phaseolina was re-isolated from symptomatic root tissues and identified by phylogenetic approach, fulfilling Koch's postulates. To date, this fungus affects at least 372 hosts globally causing yield losses. Although in Mexico this fungus has been documented in Glycine max, Ipomoea batatas, Phaseolus vulgaris, Physalis ixocarpa, Saccharum officinarum, Sesamum indicum, Solanum melongena, S. tuberosum, and Sorghum bicolor (Farr and Rossman 2021). However, there are no reports of M. phaseolina as a potential pathogen on peanut; therefore, according to our knowledge, this is the first report of this fungus affecting A. hypogaea in Mexico.
RESUMO
Heterodera glycines, the soybean cyst nematode, and Macrophomina phaseolina, causal agent of charcoal rot, are economically important soybean pathogens. The impact and effect of these pathogens on soybean yield in coinfested fields in the Midwest production region is not known. Both pathogens are soilborne, with spatially aggregated distribution and effects. Spatial regression analysis, therefore, is an appropriate method to account for the spatial dependency in either the dependent variable or regression error term from data produced in fields naturally infested with H. glycines and M. phaseolina. The objectives of this study were twofold: to evaluate the combined effect of H. glycines and M. phaseolina on soybean yield in naturally infested commercial fields with ordinary least squares and spatial regression models; and to evaluate, under environmentally controlled conditions, the combined effect of H. glycines and M. phaseolina through nematode reproduction and plant tissue fungal colonization. Six trials were conducted in fields naturally infested with H. glycines and M. phaseolina in Ohio. Systematic-grid sampling was used to determine the population densities of H. glycines and M. phaseolina, and soybean yield estimates. Though not used in any statistical analysis, M. phaseolina colony forming units from plant tissue, charcoal rot severity, and H. glycines type were also recorded and summarized. In two greenhouse experiments, treatments consisted of H. glycines alone, M. phaseolina alone, and coinfestation of soybean with both pathogens. Moran's I test indicated that the yield from five fields was spatially correlated (P < 0.05) and aggregated. In these fields, to account for spatial dependence, spatial regression models were fitted to the data. Spatial regression analyses revealed a significant interaction effect between H. glycines and M. phaseolina on soybean yield for fields with high initial population densities of both pathogens. In the greenhouse experiments, H. glycines reproduction was significantly (P < 0.05) reduced in the presence of M. phaseolina; however, soybean tissue fungal colonization was not affected by the presence of H. glycines. The direct mechanisms by which H. glycines and M. phaseolina interact were not demonstrated in this study. Future studies must be conducted in the field and greenhouse to better understand this interaction effect.
Assuntos
Glycine max , Tylenchoidea , Animais , Ohio , Doenças das Plantas , Regressão EspacialRESUMO
Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes --#91;pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase--#93; by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3 U/ml and polymethylgalacturonase between 0.15 and 1.3 U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36 U/l and 63 U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.
Macrophomina phaseolina es un fitopatógeno polífago, causante de podredumbre carbonosa. Los daños que genera en cultivos de soja y maíz bajo siembra directa en Argentina, en períodos secos y calurosos, se incrementaron por su habilidad para sobrevivir como esclerocios en suelos y restos de cosecha. El propósito del trabajo fue estudiar la producción in vitro de enzimas degradadoras de pared celular vegetal (pectinasas --#91;poligalacturonasa y polimetilgalacturonasa--#93;; celulasas --#91;endoglucanasa--#93;; hemicelulasas --#91;endoxilanasa--#93; y la enzima ligninolítica lacasa) de varios aislamientos argentinos de M. phaseolina y evaluar la patogenicidad de esos aislamientos, como paso preliminar para establecer el papel de estas enzimas en la interacción M. phaseolina-maíz. Se estudió la cinética de crecimiento del hongo y la de la producción de dichas enzimas en medios de cultivo líquidos sintéticos con ácido glutámico como fuente de nitrógeno y con pectina, carboximetilcelulosa (CMC) o xilano como fuentes de carbono. Las pectinasas fueron las primeras enzimas detectadas y los máximos títulos registrados (1,4 UE/ml --#91;poligalacturonasa--#93; y 1,2 UE/ml --#91;polimetilgalacturonasa--#93;, respectivamente) superaron a los de celulasas y xilanasas, que aparecieron más tardíamente y en menor magnitud. Esta secuencia promovería la maceración inicial del tejido, seguida luego por la degradación de la pared celular vegetal. Se detectó actividad lacasa en todos los aislamientos (36 a 63 U/l). La agresividad de todos los aislamientos resultó alta en los 3 hospedantes evaluados: semillas de maíz, de girasol y de melón. En este trabajo se investiga por primera vez el potencial de distintos aislamientos de M. phaseolina para producir enzimas degradadoras de pared celular vegetal en cultivo líquido.
Assuntos
Técnicas In Vitro/métodos , Parede Celular/enzimologia , Zea mays/enzimologia , Zea mays/parasitologia , Poligalacturonase/isolamento & purificação , Celulase/isolamento & purificação , Endo-1,4-beta-Xilanases/isolamento & purificaçãoRESUMO
El hongo Macrophomina phaseolina (Tassi) Goid., agente causal de la enfermedad denominada «pudrición carbonosa¼, provoca pérdidas significativas en la producción de cultivos como maíz, sorgo, soya y frijol en México. Este hongo, parásito facultativo, muestra amplia capacidad de adaptación a ambientes estresantes, donde existen altas temperaturas y deficiencia hídrica, condiciones frecuentes en gran parte de la agricultura de dicho país. En este trabajo se describen algunos aspectos básicos de la etiología y la epidemiología de M. phaseolina. Asimismo, se revisa la importancia que guardan las respuestas de este hongo a ambientes estresantes, particularmente la deficiencia hídrica, de acuerdo con caracteres morfológicos y del crecimiento, así como fisiológicos, bioquímicos y de patogenicidad. Finalmente, se presentan algunas perspectivas de estudio del género, que enfatizan la necesidad de mejorar su conocimiento, con base en la aplicación de herramientas tradicionales y de biotecnología, y de dilucidar mecanismos de tolerancia al estrés ambiental, extrapolables a otros microorganismos útiles al hombre.
Fungus Macrophomina phaseolina (Tassi) Goid. is the causative agent of charcoal rot disease which causes significant yield losses in major crops such as maize, sorghum, soybean and common beans in Mexico. This fungus is a facultative parasite which shows broad ability to adapt itself to stressed environments where water deficits and/or high temperature stresses commonly occur. These environmental conditions are common for most cultivable lands throughout Mexico. Here we describe some basic facts related to the etiology and epidemiology of the fungus as well as to the importance of responses to stressed environments, particularly to water deficits, based on morphology and growth traits, as well as on physiology, biochemistry and pathogenicity of fungus M. phaseolina. To conclude, we show some perspectives related to future research into the genus, which emphasize the increasing need to improve the knowledge based on the application of both traditional and biotechnological tools in order to elucidate the mechanisms of resistance to environmental stress which can be extrapolated to other useful organisms to man.
Assuntos
Adaptação a Desastres , Meio Ambiente , Produção Agrícola/economia , Fungos/crescimento & desenvolvimento , Fungos/fisiologia , Fungos/patogenicidade , Estresse Fisiológico/fisiologiaRESUMO
Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Argentina , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , Carbono/metabolismo , Parede Celular/metabolismo , Celulase/isolamento & purificação , Celulase/metabolismo , Citrullus/microbiologia , Meios de Cultura , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Helianthus/microbiologia , Microbiologia Industrial/métodos , Lacase/isolamento & purificação , Lacase/metabolismo , Nitrogênio/metabolismo , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Sementes/microbiologia , Zea mays/microbiologiaRESUMO
Fungus Macrophomina phaseolina (Tassi) Goid. is the causative agent of charcoal rot disease which causes significant yield losses in major crops such as maize, sorghum, soybean and common beans in Mexico. This fungus is a facultative parasite which shows broad ability to adapt itself to stressed environments where water deficits and/or high temperature stresses commonly occur. These environmental conditions are common for most cultivable lands throughout Mexico. Here we describe some basic facts related to the etiology and epidemiology of the fungus as well as to the importance of responses to stressed environments, particularly to water deficits, based on morphology and growth traits, as well as on physiology, biochemistry and pathogenicity of fungus M. phaseolina. To conclude, we show some perspectives related to future research into the genus, which emphasize the increasing need to improve the knowledge based on the application of both traditional and biotechnological tools in order to elucidate the mechanisms of resistance to environmental stress which can be extrapolated to other useful organisms to man.