First Confirmed Report of Rhizoctonia solani AG-7 Causing Potato Stem Canker in Mexico.

Rhizoctonia solani, is the causal agent of black scurf and stem canker of potatoes (Solanum tuberosum L.) throughout the world. In November 2021, stem canker symptoms were observed in two potato fields located in Ahome, Sinaloa, Mexico. The disease incidence was estimated up to 15%. For fungal isolation, fragments of symptomatic stems were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed with sterilized distilled water, and blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25°C in darkness for 4 days. Rhizoctonia-like colonies were consistently obtained and 12 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially and then turned brown, raised, and with entire or undulate edges. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by staining with 1% safranin O and 3% KOH solution showed multinucleate cells. The morphological features of the isolates resembled those of Rhizoctonia solani (Sneh et al. 1991). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF267, CCLF274, CCLF277, and CCLF279. For molecular identification, genomic DNA from each of the four isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP784258 to OP784261). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the four isolates within the R. solani AG-7 clade with high bootstrap support (100%). For pathogenicity tests, certified pathogen-free potato mini-tuber (cv. Fianna) were placed in a polystyrene pot (1 L) filled with a 5 cm layer of a sterile substrate composed of soil and peat moss (2:1 w/w). One rice grain (20 mg) colonized with each isolate was placed 10 mm above the uppermost sprout tip and covered with the sterile substrate (Inokuti et al. 2019). Control plants were inoculated with sterile rice grains. All pots were transferred to a greenhouse where the temperature ranged from 20 to 32°C. Stem necrosis symptoms were observed on all inoculated plants 25 days after emergence, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected stems and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. The AG-7 has been previously reported to cause potato diseases in South Africa (Truter and Wehner 2004). In Mexico, Carling et al. (1998) reported the presence of an isolate of R. solani AG-7 obtained from a potato tuber-borne sclerotium in Toluca; however, there is no information about the methodology used for the characterization of that isolate. To our knowledge, this is the first confirmed report of R. solani AG-7 causing potato stem canker in Mexico. Our findings improve knowledge about R. solani AGs occurring in potato fields in Mexico. So, further studies should be conducted to investigate the diversity, prevalence, and fungicide sensitivity of AGs distributed in the main potato-producing states in Mexico.

First Report of Lasiodiplodia brasiliensis Causing Root Rot on Watermelon in Brazil.

Watermelon (Citrullus lanatus (Thumb.) Matsum. & Nakai) is an important crop in Brazil both for export and domestic consumption. In October 2019, watermelon plants showing decline and root rot symptoms were surveyed in 16 commercial fields in Baraúna's municipality (Rio Grande do Norte, Brazil). The disease prevalence was 12.5%, and the average incidence was 5%. Affected root segments were cut into small pieces and surface-disinfected with 70% ethyl alcohol and 1.5 % NaOCl for 1 and 2 min, respectively. Tissues were plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fungal colonies developed from the infected tissues were dark or greyish, and pure cultures were obtained by hyphal tip isolation technique. Six fungal isolates with the same morphology were obtained. Two of them were selected for morphological and molecular characterization (CFC-1123 and CFC-1124). Isolates grew rapidly in PDA, covering the entire surface of the Petri dishes within 3 days. The aerial mycelium was initially white, turning dark greenish-gray after 4 to 5 days of incubation at 25°C in the dark. Isolates produced pycnidia and conidia in water-agar medium with sterilized pine needles after 30 days of incubation at 25°C under near-UV light. The conidia were initially hyaline and brown with central transverse septum and longitudinal streaks when mature. Conidia were ellipsoid to oval (22.83 ± 3.1 µm long and 11.58 ± 1.5 µm wide). Based on morphological features, the isolates were initially identified as Lasiodiplodia sp. (Phillips et al. 2013). To confirm the identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region as well as fragments of the translation elongation factor 1-α (TEF) and ß-tubulin 2 (TUB) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank under accession numbers OL841380, OL865376 and OL890691 for CFC-1123, and OL841381, OL865377 and OL890692 for CFC-1124. Maximum likelihood phylogenetic analysis of the concatenated sequences of ITS, TEF and TUB gene regions of some reference sequences and ex-types of Lasiodiplodia spp. was performed. Phylogenetic analysis revealed that the isolates grouped in the L. brasiliensis clade (Netto et al. 2014) with 80/79% of bootstrap. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi from Cariri (CFC) at the Universidade Federal do Cariri (Crato, Brazil). Pathogenicity of the two isolates was determined using colonized wheat grains as inoculum source. One watermelon seed (cv. Crimson Sweet) was placed in a sterile plastic pot (500-mL) filled with 6 cm layer of a substrate composed of soil and Tropstrato® (5:1 w/w). Three wheat grains (50 mg) colonized with each isolate were placed 10 mm above the seed and covered with the substrate. Control pots were inoculated only with sterile wheat grains. There were five replicates for each isolate. The pots with seedlings were maintained in a greenhouse at 28 ± 2°C under natural light conditions. The inoculated seedlings showed poor growth, withering and drying leaves 45 days after inoculation (DAI), and subsequently root rot symptoms and death at 60 DAI. Control seedlings remained asymptomatic. The pathogen was re-isolated from all inoculated seedlings and identified by conidia morphology to fulfill Koch's postulates. Lasiodiplodia brasiliensis has been reported to cause postharvest rot and gummosis of watermelon (Farr and Rossman 2022). However, to our knowledge, this is the first report of watermelon decline caused by this fungus in Brazil and worldwide. This finding must be considered for developing efficient control strategies for the disease.

Characterization of difenoconazole resistance in Lasiodiplodia theobromae from papaya in Brazil.

BACKGROUND: Stem-end rot caused by Lasiodiplodia theobromae is one of the most important diseases of papaya in northeastern Brazil. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but the occurrence of DMI resistance may decrease fungicide efficacy. RESULTS: Detached fruit studies revealed that isolates with EC50 values of 6.07 and 6.28 µg mL-1 were not controlled effectively, but reduced virulence and ability to grow at temperatures ranging from 12 to 32 °C suggesting fitness penalties were observed. Cross-resistance was observed only between difenoconazole and propiconazole. The entire cytochrome P450 sterol 14α-demethylase (LtCYP51) gene and its flanking regions were cloned. The gene was 1746 bp in length and contained three introns. The predicted protein contained 525 amino acids. Phylogenetic tree analysis showed that the LtCYP51 belongs to the CYP51B clade. No amino acid variation was found between sensitive and resistant isolates; however, the gene was constitutively more highly expressed in resistant isolates. CONCLUSION: Resistance to DMI fungicides in L. theobromae is based on LtCYP51 gene overexpression and fitness penalties may be present in difenoconazole-resistant isolates. © 2019 Society of Chemical Industry.

Reduced sensitivity of azoxystrobin and thiophanate-methyl resistance in Lasiodiplodia theobromae from papaya.

Stem-end rot caused by Lasiodiplodia theobromae is one of the most devastating diseases of papaya in northeastern Brazil. It is most effectively controlled by applications of fungicides, including site-specific fungicides at risk for resistance development. This study investigated the molecular mechanisms of reduced sensitivity to the QoI fungicide azoxystrobin and resistance to the MBC fungicide thiophanate-methyl in L. theobromae from Brazilian orchards. The EC50 values for azoxystrobin in sixty-four isolates ranged from 0.36 µg/ml to 364.24 µg/ml and the frequency distribution of EC50 values formed a multimodal curve, indicating reduced sensitivity to azoxystrobin. In detached fruit assays reduced sensitive isolates were not controlled as effectively as sensitive isolates at lowest label rate. Partial fragments were obtained from target genes ß-tubulin (751 bp) and Cytb (687 bp) of isolates resistant to thiophanate-methyl and reduced sensitivity to azoxystrobin. Sequence analysis of the ß-tubulin fragment revealed a mutation corresponding to E198K in all thiophanate-methyl-resistant isolates, while reduced sensitivity to axoxystrobin was not attributable to Cytb gene alterations. The target gene-based mechanism conferring resistance to thiophanate-methyl will likely be stable even if selection pressure subsides. However, the mechanism conferring reduced sensitivity to azoxystrobin is not based on target gene modifications and thus may not be as stable as other genotypes with mutations in Cytb gene.

Thiophanate-Methyl Resistance and Fitness Components of Colletotrichum musae Isolates from Banana in Brazil.

Anthracnose, caused by Colletotrichum musae, is the most important postharvest disease of banana and is widely distributed among the banana production regions in Brazil. Although thiophanate-methyl is a fungicide frequently used in Brazilian banana orchards to control Sigatoka leaf spot, Collettotrichum populations are also exposed, resulting in the evolution of fungicide resistance and the inability to manage banana anthracnose. We investigated 139 Brazilian isolates of C. musae for thiophanate-methyl sensitivity in vitro. The 50% mycelial growth inhibition (EC50) values varied between 0.003 and 48.73 µg/ml. One-hundred and thirty isolates were classified as sensitive, with EC50 values ranging from 0.003 to 4.84 µg/ml, while the remaining nine isolates were considered moderately resistant, with EC50 values ranging between 10.43 and 48.73 µg/ml. Resistant or highly resistant isolates (EC50 > 100 µg/ml) were not found. A substitution of TAC for TTC at codon 200 in a coding region of the ß-tubulin gene was associated with the moderately resistant phenotype. Applications of thiophanate-methyl formulation to detached banana fruit at the label rate (500 µg/ml) showed low efficacy in controlling the moderately resistant isolates on banana fruits. However, there is no indication of a reduction in fitness associated with fungicide resistance as sensitive and moderately resistant isolates do not differ with respect to mycelial growth rate (P = 0.098), spore production (P = 0.066), spore germination (P = 0.366), osmotic sensitivity (P = 0.051), and virulence (P = 0.057). Our results revealed absence of adaptability cost for the moderately resistant isolates, suggesting that they can be dominant in population if the fungicide continue to be applied.

Escala diagramática para avaliação da severidade da antracnose em pinha / Diagrammatic scale for anthracnose assessment severity in custard apple

A antracnose, causada por Colletotrichum gloeosporioides, é uma importante doença da pinha (Annona squamosa) no Brasil e em nível mundial. Devido à inexistência de métodos padronizados para quantificação dessa doença, foi desenvolvida uma escala diagramática com níveis de 1, 2, 5, 10, 20 e 40 por cento de área foliar lesionada. A escala diagramática foi validada por 10 avaliadores, baseado em 48 folhas de pinha com diferentes níveis de severidade da antracnose, mensurados previamente com o programa Assess®. A acurácia, a precisão e a reprodutibilidade das estimativas de cada avaliador foi determinada por regressão linear simples entre a severidade real e a estimada, com e sem o auxílio da escala. Com a escala, os avaliadores obtiveram melhores níveis de acurácia e precisão das estimativas, com os erros absolutos concentrando-se na faixa de 10 por cento. Os avaliadores apresentaram elevada repetibilidade (91 por cento) e reprodutibilidade (³90 por cento em 94,5 por cento dos casos) das estimativas com a utilização da escala. Portanto, a escala diagramática proposta é adequada para avaliação da severidade da antranose da pinha.

Anthracnose caused by Colletotrichum gloeosporioides is an important disease of custard apple (Annona squamosa) in Brazil and worldwide. Due to the inexistence of standard methods for the assessment of that disease, a diagrammatic scale was developed with 1, 2, 5, 10, 20 and 40 percent of diseased leaf area. The diagrammatic scale was validated by 10 raters using 48 custard apple leaves with different levels of anthracnose severity previously measured by the software Assess®. The accuracy, precision and reproducibility estimative of each rater were determined by simple linear regression between actual and estimated severity, with and without the use of the scale. With the scale, the raters obtained better levels of accuracy and precision, with absolute errors concentrating around 10 percent. Raters showed high repeatability (91 percent) and reproducibility (³90 percent in 94.5 percent of the cases) of the estimates by using the scale. The proposed diagrammatic scale is suitable for the evaluation of anthracnose severity in custard apple.

Amostragem, caracterização de sintomas e escala diagramática da mancha graxa dos citros (Mycosphaerella citri) no Recôncavo Baiano / Sampling, characterization of citrus greasy spot (Mycosphaerella citri) symptoms in Recôncavo Baiano and development of a diagrammatic scale for assessment of severity

O Recôncavo da Bahia é uma região de clima propício à mancha graxa dos citros (Mycosphaerella citri), doença nunca antes estudada no Brasil. Os objetivos deste trabalho foram definir o tamanho mínimo de amostra para quantificar a incidência, caracterizar os sintomas da doença e elaborar uma escala diagramática para a avaliação de sua severidade. A partir de uma amostragem piloto realizada por avaliação de cinco folhas por quadrante, quatro quadrantes por planta e 30 plantas em cada um dos 10 pomares visitados no município de Cruz das Almas, foi determinado que o tamanho mínimo da amostra em quadrantes e folhas seria de quatro e 16 plantas, respectivamente. De 320 folhas coletadas de diferentes plantas em 11 pomares, foi constatada uma grande quantidade de lesões (média de 131) por folha sintomática, em sua maioria diminutas (média de 0,014cm²). A severidade variou de 0,15 por cento a 35,85 por cento, com média de 7,3 por cento. Com base na severidade real em campo, foi elaborada uma escala diagramática com seis níveis: 1 por cento, 2 por cento, 5 por cento, 9 por cento, 18 por cento e 36 por cento de área foliar lesionada. Cinqüenta imagens de folhas foram submetidas a avaliadores por duas vezes, os quais estimaram a severidade com e sem a utilização da escala, constatando-se um discreto benefício no grau de precisão e de acurácia das estimativas com o uso da escala.

Weather conditions make the Recôncavo Region in the State of Bahia, a favorable region to the occurrence of citrus greasy spot (Mycosphaerella citri), a disease that hadn't been studied before in Brazil. Thus, this research aimed to develop a sampling method to quantify its incidence, to characterize its symptoms, and to develop a diagrammatic scale for assessing disease severity. The minimum sample size to estimate greasy spot incidence in leaves (16 plants) was calculated based on a prospective sampling of five leaves per quadrant, four quadrants per tree, and 30 trees per grove, in ten groves. From 320 mature leaves collected in different plants of 11 groves, it was observed a high number of lesions per symptomatic leaf (mean of 131±102), most of them small (mean of 0.014±.,011cm²). Greasy spot severity varied from 0,15 percent to 35,85 percent, with a mean of 7,3 percent. Based on those severity values, a six level (1 percent, 2 percent, 5 percent, 9 percent, 18 percent e 36 percent of lesioned leaf area) diagrammatic scale was developed. 50 images of symptomatic leaves were showed to evaluators, which estimated the severity with and without the use of the scale. The diagrammatic scale use, slightly increased the estimates accuracy and precision.

Escala diagramática para avaliação da severidade da cercosporiose em caupi / Diagrammatic scale for assessment of Cercospora leaf spot severity in cowpea

A cercosporiose, causada por Cercospora cannescens e Pseudocercospora cruenta, é uma importante doença do caupi (Vigna unguiculata) no Brasil. Devido à inexistência de métodos padronizados para quantificação dessa doença, foi desenvolvida uma escala diagramática com níveis de 1, 2, 4, 8, 16, 32, 64 e 82 por cento de área foliar lesionada. A escala diagramática foi validada por 10 avaliadores, usando 50 folíolos de caupi com diferentes níveis de severidade, mensurados previamente com o programa Assess®. A acurácia, a precisão e a reprodutibilidade das estimativas de cada avaliador foi determinada por regressão linear simples entre a severidade real e a estimada, com e sem o auxílio da escala. Com a escala, os avaliadores obtiveram melhores níveis de acurácia e precisão das estimativas, com os erros absolutos concentrando-se na faixa de 10 por cento. Os avaliadores apresentaram elevada repetibilidade (94 por cento) e reprodutibilidade (90 por cento em 82,3 por cento dos casos) das estimativas com a utilização da escala. Portanto, a escala diagramática proposta é adequada para avaliação da severidade da cercosporiose do caupi.

Cercospora leaf spot caused by Cercospora canescens and Pseudocercospora cruenta is an important disease of cowpea (Vigna unguiculata) in Brazil. Due to the inexistence of standard methods for the assessment of this disease, a diagrammatic scale was developed with 1, 2, 4, 8, 16, 32, 64 and 82 percent of diseased leaf area. The diagrammatic scale was validated by 10 raters using 50 cowpea leaflets with different levels of severity previously measured by the software Assess®. The accuracy, precision and reproducibility of the estimative of each rater were determined by simple linear regression between actual and estimated severity, with and without the use of the scale. With the scale raters obtained better levels of accuracy and precision, with absolute errors concentrating around 10 percent. Raters showed high repeatability (94 percent) and reproducibility ( 90 percent in 82.3 percent of the cases) of the estimates by using the scale. The proposed diagrammatic key is suitable for the evaluation of Cercospora leaf spot severity in cowpea.

Escala diagramática para avaliação da severidade da antracnose em bastão do imperador / Diagrammatic scale for assessment of torch ginger anthracnose severity

A antracnose, causada pelo fungo Colletotrichum gloeosporioides (Penz.) Sacc., é uma importante doença do bastão do imperador [Etlingera elatior (Jack) Smith] no Nordeste brasileiro. Considerando a inexistência de métodos padronizados para quantificação dessa doença, foi elaborada uma escala diagramática com os níveis 1, 2, 4, 8, 16, 32, 64, 82 e 92 por cento de área da bráctea lesionada, testando-se a acurácia, a precisão e a reprodutibilidade das estimativas de severidade da antracnose com e sem a utilização da escala. A escala diagramática foi validada por 10 avaliadores, usando 45 brácteas com diferentes níveis de severidade da antracnose. Sem a escala, a maioria dos avaliadores superestimou a severidade da doença. Com a escala, os avaliadores obtiveram melhores níveis de acurácia e precisão, com os erros absolutos concentrando-se na faixa de 10 por cento. Os avaliadores apresentaram elevada repetibilidade (95 por cento) e reprodutibilidade (³90 por cento em 75,6 por cento dos casos) das estimativas com a utilização da escala. A escala diagramática proposta demonstrou ser adequada para avaliação da severidade da antracnose em bastão do imperador.

Anthracnose caused by the fungus Colletotrichum gloeosporioides (Penz.) Sacc. is an important disease of torch ginger [Etlingera elatior (Jack) Smith] in the Brazilian Northeast. Considering the inexistence of standard methods for assessment of this disease, a diagrammatic scale was developed with 1, 2, 4, 8, 16, 32, 64, 82 and 92 percent of diseased bracts area and tested for accuracy, precision and reproducibility of the estimative of anthracnose severity with and without using the scale. The diagrammatic key was validated by 10 raters, using 45 bracts with different levels of severity. Without the diagrammatic scale most of raters overestimated disease severity. With the scale raters obtained better levels of accuracy and precision, with absolute errors concentrated around 10 percent. Raters showed great repeatability (95 percent) and reproducibility (³90 percent in 75.6 percent of cases) of estimative by using the scale. The proposed diagrammatic scale was suitable for the evaluation of torch ginger anthracnose severity.

Sobrevivência saprofítica de Alternaria brassicicola e manejo de restos foliares de brócolos / Saprophytic survival of Alternaria brassicicola and management of broccoli leaf debris

A sobrevivência saprofítica do fungo Alternaria brassicicola foi investigada em restos foliares de brócolos (Brassica oleracea var. italica), em diferentes profundidades de incorporação no solo, períodos do ano e sistemas de manejo do solo. Os restos foliares infectados pelo patógeno foram distribuídos em parcelas no campo, na superfície do solo e nas profundidades de 5 e 10cm. Periodicamente, os restos foliares foram coletados e a concentração de conídios quantificada, sendo obtida a longevidade da esporulação e a taxa de extinção da esporulação. Foi verificada maior longevidade da esporulação nos restos foliares no período com temperaturas amenas, maior umidade relativa do ar e menor precipitação pluvial. Menor esporulação ocorreu quando os restos culturais foram incorporados a 10cm de profundidade. Quando comparados os sistemas de manejo convencional e orgânico, houve diferença na taxa de extinção da esporulação do patógeno quando a fonte de inóculo foi depositada nas profundidades de 5 e 10cm, sendo superior no solo sob manejo convencional, enquanto na superfície do solo não houve diferença. O manejo da alternariose em brócolos na região do estudo pode ser realizado pela incorporação dos restos foliares infectados no solo, à profundidade mínima de 10cm, visando a um intervalo mínimo de 60 dias entre cultivos de brássicas.

The saprophytic survival of the fungus Alternaria brassicicola was investigated in leaf debris of broccoli (Brassica oleracea var. italica), at different depths of soil incorporation, periods of the year, and systems of soil management. Infected leaf debris were distributed in field plots, at the soil surface and at depths of 5 and 10cm. Periodically, the debris in the bags were collected and conidia concentrations were quantified for longevity of spore production and extinction rate. Higher spore production longevity was observed on leaf debris in the period with lower temperatures, higher air relative humidity and lower rainfall. The lowest spore production occurred when leaf debris incorporation was made at 10cm depth. In relation to soil management systems, when the inoculum source was deposited at the soil surface no differences in the rate of spore production were observed, but at depths of 5 and 10cm, the rate was significantly higher in the soil under conventional management. The management of Alternaria black spot on broccoli at the area under study can be accomplished by incorporating infected leaf debris in the soil, at least 10cm depth, for no less than a 60-day interval in subsequent brassica plantings.

Reacão de clones de videira a Xanthomonas campestris pv. viticola, baseada nos componentes epidemiológicos do cancro bacteriano / Reaction of grapevine clones to Xanthomonas campestris pv. viticola, based on epidemiological components of bacterial canker

O cancro bacteriano, causado por Xanthomonas campestris pv. viticola (Xcv), é a doenca bacteriana mais importante da videira na região do Submédio São Francisco. A reacão de 20 clones de videira, sendo 13 de copa e sete de porta-enxerto, foi avaliada quanto à resistência ao patógeno, em casa de vegetacão. As plantas foram inoculadas com a suspensão do isolado Xcv1 (A570=108UFC mL-1), incubadas em casa de vegetacão e observadas diariamente quanto aos componentes epidemiológicos do cancro bacteriano: período de incubacão, incidência de folhas com sintomas, incidência de folhas com cancro, severidade da doenca, taxa de progresso da incidência da doenca, área abaixo da curva de progresso da severidade da doenca. Todos os clones foram suscetíveis ao patógeno, embora diferindo significativamente entre si (P=0,05) para a maioria das variáveis analisadas. Em geral, 'Brasil' apresentou os maiores níveis de doenca para todas as variáveis testadas, enquanto'Isabel' e 'Paulsen 1103' destacaram-se ao propiciarem os maiores valores de período de incubacão e os menores valores de incidência de folhas com sintomas, com cancros, severidade da doenca, taxa de progresso de incidência da doenca e área abaixo da curva de progresso da severidade da doenca, indicando grande potencial de utilizacão em programas de melhoramento genético e manejo integrado. As correlacões significativas (P=0,05) verificadas entre as variáveis estudadas indicam que qualquer delas pode ser utilizada em pesquisas envolvendo reacão de clones ao cancro bacteriano da videira. Quando considerado o conjunto dos componentes epidemiológicos, o agrupamento pelo método UPGMA (agrupamento aos pares pela média aritmética não ponderada) permitiu a separacão dos clones de copa e porta-enxerto em três grupos de similaridade cada.