Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Abstract Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Resumo A ferrugem das folhas de maydis, causada por Bipolaris maydis, é uma doença importante da cultura do milho em Khyber Pakhtunkhwa (KP), Paquistão. Quinze isolados do patógeno, coletados em KP, foram estudados quanto à variabilidade com base em marcadores fenotípicos e moleculares. Variabilidade significativa entre os isolados foi observada quando avaliada por meio de características fenotípicas, como crescimento radial, concentração de esporos, sensibilidade a fungicida e virulência. Os isolados foram classificados em seis grupos de cultura com base na cor, textura e margens da colônia. A morfologia dos conídios também foi variável. Estes eram retos ou ligeiramente curvos e de cor marrom-claro a escuro. O teste de fungicida mostrou variação significativa no grau de sensibilidade ao carbendazim. O isolado Bm8 exibiu crescimento radial máximo em placas com adição de carbendazim. Por outro lado, o isolado Bm15 apresentou o menor crescimento radial. As variações no padrão de virulência dos isolados foram evidentes quando uma variedade de milho suscetível Azam foi inoculada com esporos de B. maydis. A variabilidade genética entre os isolados também foi estimada por RAPD, bem como sequenciamento da região ITS. O dendrograma RAPD agrupou todos os isolados em dois grupos principais. A distância genética média variou de 0,6% a 100%, indicando uma lacuna genética diversa entre os isolados. A distância genética máxima foi encontrada entre os isolados Bm9 e Bm10 e também entre Bm2 e Bm8. Por outro lado, os isolados Bm13 e Bm15 estavam a uma distância genética mínima. O dendrograma filogenético baseado no sequenciamento da região ITS agrupou todos os isolados em um único aglomerado principal. Os agrupamentos em ambos os dendrogramas não se correlacionam com a distribuição geográfica nem com as características morfológicas.

Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach / Introdução do gene resistente à ferragem da folha bacteriana (BLB), xa7 na variedade MARDI elite, MR219 por abordagem de backcrossing assistida por marcador (MABC)

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

First Report of Pantoea ananatis and Pantoea eucalypti causing onion leaf blight and bulb decay in Central Chile.

Nearly 5,400 hectares of long-day onions (Allium cepa) are cultivated in the Central Zone of Chile (UTM 33°3´S to 37°24´S). During summer 2021-22 and 2022-23 (December to February), followed by high temperatures (around 35°C) and high humidity conditions, symptoms showing yellowing, soft rot, blight on old leaves, necrotic tips, and soft bulbs were observed. The affected plants were observed in "spots" into the fields, and the incidence reached 5-10%. The severity was high, and 70-80% of the affected plants died. Symptomatic plants from different fields from the Coquimbo, Metropolitan, O´Higgins and Maule regions were sampled. Isolations were made using casamino-acid peptone glucose (CPG) agar medium (Schaad, 2001). Yellow-pigmented, circular to irregular shaped colonies were observed. Molecular identification was carried out by partial 16S rRNA gene sequencing, resulting in the identification of 18 isolates of Pantoea spp. from twelve different fields. Selected strains were biochemically analyzed using the GEN III BIOLOG microtest system (Hayward, CA) and were identified as Pantoea spp. BLAST analyses of the 16S rRNA sequences (602 nt) of selected strains (GenBank Accession No. OR527817 to OR527819) against the NCBI Database resulted in the identification of Pantoea species with 100% coverage and 100% identity. To determine the Pantoea species of each strain, housekeeping gene gyrB (Delétoile et al. 2009) was amplified and sequenced (GenBank Accessions No. OR544061 to OR544063). BLAST analysis (802 nt) of selected strains resulted in 100% coverage and 100% identity, identifying three different species: P. ananatis, P. eucalypti, and P. agglomerans. Pantoea species were isolated from both leaves and bulbs and no more than one species was observed per field. Pathogenicity assays in onion plants and bulbs were performed based on the methodology described by Asselin et al. (2018). Five onion plants cv. Cimarron of thirteen-week-old were inoculated by wounding an external leave with a sterile toothpick previously immersed in a bacterial suspension at ~ 108 CFU/mL and maintained at 26-28°C for 30 days in high humidity conditions. Control plants were inoculated with sterile water. Plants inoculated with Pantoea spp. showed chlorosis, soft rot, and necrosis mainly in older leaves, as observed in the field, while negative control plants remained healthy. Pantoea spp. were re-isolated from the inoculated onion plants. Toothpicks dipped in the inoculum were stuck 4 cm into the shoulders of onion bulbs and incubated at 26°C for 20 days. Water was inoculated as a negative control. At the end of the incubation period, the bulbs were opened longitudinally across their inoculation sites and shrunken, brownish, watery scales were observed. Pantoea species have been previously described as causing leaf blight of onions in Georgia and Michigan in the USA and South Africa, Brazil, and Uruguay (Hattingh and Walters 1981; Gitaitis and Gay 1997; Edens et al. 2006; Tho et al. 2015; De Armas et al. 2022; Rosende et al. 2022). P. agglomerans was recently reported in Chile (Sepúlveda et al. 2023), but this is the first report of P. ananatis and P. eucalypti affecting onions in central Chile. This detection is an alert call for the onion's growers and exporters in Chile for upcoming seasons, where conditions predisposing to disease may continue to occur. It is crucial to continue analyzing the factors that caused the appearance of this new disease in onions.

Performance- and Resistance-Related Early Responses of Colombian Elite Rubber Tree Genotypes under Low Pressure of South American Leaf Blight: Implications for Disease Management in the Amazon.

The cultivation of Hevea brasiliensis, the primary commercial source of natural rubber, is strongly impacted by South American leaf blight (SALB) disease, caused by the fungus Pseudocercospora ulei. Various management strategies have been implemented, including the selection of resistant genotypes and the identification of escape zones. This study evaluated the growth, early yield, and resistance to SALB of nine Colombian elite genotypes from the ECC-100 series and IAN 873 clone (control) in a large-scale clone trial in an area with low SALB pressure in the Colombian Amazon during 2017-2020. Favorable early performance was evident, although there was a significant increase in the severity and sporulation of P. ulei over time, especially in the ECC 35, ECC 60, and IAN 873 genotypes. However, these scores indicate low susceptibility. Genotypes with higher resistance to SALB demonstrated greater growth and early yield compared to more highly susceptible genotypes. The ECC 64, ECC 73, ECC 90, ECC 25, and ECC 29 genotypes were more desirable in low SALB pressure zones due to their higher resistance and early performance. It is important to highlight that this research contributes to the selection of new SALB-resistant Colombian genotypes of H. brasiliensis. However, it is also necessary to evaluate the productivity of these selections in the mature stage and long-term resistance to SALB before recommending and promoting their commercial adoption in the Colombian Amazon.

Occurrence and Distribution of Physiological Races of Exserohilum turcicum in Maize-Growing Regions of Mexico.

Turcicum leaf blight (TLB) is a common foliar disease of maize in Mexico that is caused by the fungal pathogen Exserohilum turcicum. The most effective management strategy against TLB is monogenic race-specific resistance. Among the 140 E. turcicum isolates from symptomatic leaves collected from maize fields in Mexico, 100 were obtained from tropical (Veracruz) and temperate areas (Estado de México) between 2010 and 2019, and 40 isolates were obtained from tropical (Sinaloa, Tamaulipas, Veracruz, and Chiapas), subtropical (Nayarit, Jalisco, and Guanajuato), and temperate areas (Estado de Mexico, Hidalgo, and Puebla) collected in 2019. All the isolates caused TLB symptoms on the positive control (ht4), showing that they were all pathogenic. Six physiological races of E. turcicum (2, 3, 23, 3N, 23N, and 123N) were identified based on resistant or susceptible responses displayed by five maize differential genotypes (A619Ht1, A619Ht2, A619Ht3, B68HtN, and A619ht4). The most common was race 23, accounting for 68% of the isolates, followed by races 23N, 123N, 3, 2, and 3N at 15, 8, 6, 2, and 1%, respectively. Race 123N was able to infect the greatest number of maize differential genotypes used in the study. Race 123N was detected in Sinaloa and Estado de México. Race 3 was detected in Nayarit and Jalisco. Race 2 was detected in Jalisco, Estado de México, and Veracruz, and race 3N was detected in Tamaulipas. Race 23 was equally dominant in the tropical, subtropical, and temperate regions, while race 123N was more common in the tropical environment, and race 23N was more common in the tropical and temperate environments. There was no evidence for shifts in the races between 2010 and 2019.

Spatial analysis of factors influencing bacterial leaf blight in rice production / Análise espacial dos fatores que influenciam a praga bacteriana foliar na produção de arroz

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight that is a major threat to rice production. Crop losses in extreme situations can reach up to75%, and millions of hectares of rice are affected each year. Management of the disease required information about the spatial distribution of BLB incidence, severity, and prevalence. In this study, major rice-growing areas of Pakistan were surveyed during 2018-2019 for disease occurrence, and thematic maps were developed using geographic information system (GIS). Results showed that Narowal district had highest percentage of disease incidence (54-69%), severity (42-44%), and prevalence (72-90%) meanwhile Jhung district had the lowest incidence (21-23%), severity (18-22%), and prevalence (45-54%). To understand the environmental factors contributing to this major rice disease, the research analyze, the spatial relationships between BLB prevalence and environmental variables. Those variables include relative humidity (RH), atmospheric pressure (A.P), minimum temperature, soil organic carbon, soil pH, and elevation, which were evaluated by using GIS-based Ordinary Least Square (OLS) spatial model. The fitted model had a coefficient of determination (R2) of 65 percent explanatory power of disease development. All environmental variables showed a general trend of positive correlation between BLB prevalence and environmental variables. The results show the potential for disease management and prediction using environmental variable and assessment.

Xanthomonas oryzae pv. oryzae (Xoo) causa o crestamento bacteriano das folhas, que é uma grande ameaça à produção de arroz. As perdas de safra em situações extremas podem chegar a 75% e a milhões de hectares de arroz são afetados a cada ano. O manejo da doença exigia informações sobre a distribuição espacial da incidência, gravidade e prevalência de BLB. Neste estudo, as principais áreas de cultivo de arroz do Paquistão foram pesquisadas durante 2018 e 2019 para ocorrência de doenças, e mapas temáticos foram desenvolvidos usando o sistema de informações geográficas (GIS). Os resultados mostraram que o distrito de Narowal teve a maior porcentagem de incidência da doença (54-69%), gravidade (42-44%) e prevalência (72-90%), enquanto o distrito de Jhung teve a menor incidência (21-23%), gravidade (18-22%) e prevalência (45-54%). Para compreender os fatores ambientais que contribuem para esta importante doença do arroz, a pesquisa analisa as relações espaciais entre a prevalência de BLB e as variáveis ​​ambientais. Essas variáveis ​​incluem umidade relativa (UR), pressão atmosférica (PA), temperatura mínima, carbono orgânico do solo, pH do solo e altitude, que sendo avaliadas a partir do modelo espacial Ordinary Least Square (OLS) baseado em GIS. O modelo ajustado teve um coeficiente de determinação (R2) de 65 por cento de poder explicativo do desenvolvimento da doença. Todas as variáveis ​​ambientais apresentaram tendência geral de correlação positiva entre prevalência de BLB e variáveis ​​ambientais. Os resultados mostram o potencial de manejo e predição de doenças usando variáveis ​​e avaliações ambientais.

Foliar Lesions Induced by Pestalotiopsis arengae in Oil Palm (O × G) in the Colombian Southwest Palm Zone.

In Colombia, plantings with the oil palm hybrid between Elaeis oleifera × Elaeis guineensis, known as O × G hybrid, have increased due to its tolerance to bud rot. Despite this, different degrees of foliar necrosis, chlorosis, and leaf blight have been reported in some cultivars; therefore, this work aimed to diagnose this problem. We visited plantation plots with palms exhibiting the mentioned symptoms and collected 21 samples of affected tissues in different disease states. The affected tissues were examined and seeded in a culture medium. Pathogenicity tests were performed and the isolates were characterized by culture and morphological and molecular features. Curvularia, Colletotrichum, Phoma, and 25 Pestalotiopsis-like fungi were isolated from the foliar lesions. In the pathogenicity tests, the symptoms observed in the field were reproduced with MFTU01-1, MFTU12, and MFTU21 isolates, which were identified at the species level through a sequence analysis of three genes (ITS, TUB2, and TEF1-α) as Pestalotiopsis arengae with an identical level of 99% based on the results of BLAST and phylogenetic tree analyses. The remaining 22 Pestalotiopsis-like non-pathogenic isolates were identified as species of Neopestalotiopsis and Pseudopestalotiopsis. The direct association of P. arengae with the disease was confirmed via molecular detection in affected tissues in 15 of 21 samples collected for this evaluation. This is the first report of P. arengae as the causal agent of foliar lesions in O × G hybrid oil palm in Colombia.

First Report of Neopestalotiopsis cubana Causing leaf blight on Ixora chinensis in Malaysia.

Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 µm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 µm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS), tubulin (TUB) and translation elongation factor 1-α (TEF) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 (ITS), OM339539 (TUB) and OM339540 (TEF). They were 99% to 100% identical to ITS(KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS, TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana. Thus, the pathogen was identified as N. cubanabased on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 106 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana, indicating that it poses potential damage to the medicinal plant Bunga Jejarum in Malaysia.

Antibacterial, anti-biofilm, and anti-virulence potential of tea tree oil against leaf blight pathogen Xanthomonas oryzae pv. oryzae instigates disease suppression.

Bacterial leaf blight (BLB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), causes major annual economic losses around the world. Inorganic copper compounds and antibiotics are conventionally used to control BLB disease. They often cause environmental pollution, contributing to adverse effects on human health. Therefore, research is now leading to the search for alternative control methods. Tea tree oil (TTO) is obtained from a traditional medicinal plant, Melaleuca alternifolia, with antibacterial properties. In this study, we found that TTO showed antibacterial activity against Xoo with a minimum inhibitory concentration (MIC) of 18 mg/ml. These antagonistic activities were not limited only to planktonic cells, as further studies have shown that TTO effectively eradicated sessile cells of Xoo in both initial and mature biofilms. Furthermore, it was also observed that TTO reduced various key virulence properties of Xoo, such as swimming, swarming motility, and the production of extracellular polymeric substances, xanthomonadin, and exoenzymes. TTO triggered ROS generation with cell membrane damage as an antibacterial mode of action against Xoo. The in silico study revealed that 1,8-cineole of TTO was effectively bound to two essential proteins, phosphoglucomutase and peptide deformylase, responsible for the synthesis of EPS and bacterial survival, respectively. These antibacterial and anti-virulence activities of TTO against Xoo were further confirmed by an ex vivo virulence assay where TTO significantly reduced the lesion length caused by Xoo on rice leaves. All these data concluded that TTO could be a safe, environment-friendly alternative approach for the comprehensive management of BLB disease.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

Identification and Quantification of a Phytotoxic Metabolite from Alternaria dauci.

Alternaria dauci is the causal agent of Alternaria leaf blight (ALB) in carrot (Daucus carota) crops around the world. However, to date, A. dauci has received limited attention in its production of phytotoxic metabolites. In this investigation, the bioassay-guided isolation of the extract from liquid cultures of A. dauci resulted in the isolation of two metabolites identified as α-acetylorcinol (1) and p-hydroxybenzoic acid (2), based on their spectroscopic data and results from chemical correlation reactions. Testing of both metabolites in different assays showed an important phytotoxic activity for p-hydroxybenzoic acid (2) when tested in the leaf-spot assay on parsley (Petroselinum crispum), in the leaf infiltration assay on tobacco (Nicotiana alata) and marigold (Tagetes erecta), and in the immersion assay on parsley and parsnip (Pastinaca sativa) leaves. Quantification of the two metabolites in the crude extract of A. dauci kept at different times showed that p-hydroxybenzoic acid (2) is one of the first metabolites to be synthesized by the pathogen, suggesting that this salicylic acid derivative could play an important role in the pathogenicity of the fungus.

Calonectria pentaseptata Causes Severe Leaf Disease of Cultivated Eucalyptus on the Leizhou Peninsula of Southern China.

Eucalyptus (Myrtaceae, Myrtales) trees are widely cultivated for commercial purposes worldwide. Calonectria leaf blight is one of the most prominent diseases associated with Eucalyptus trees grown in plantations in Asia and South America. Recently, symptoms of leaf blight, shoot blight, tree death, and seedling rot caused by Calonectria species have been observed in commercial Eucalyptus plantations and nurseries in Leizhou Peninsula, which is one of the most densely Eucalyptus-planted areas in southern China. Disease samples were collected from 10 Eucalyptus species and a number of Eucalyptus grandis, E. tereticornis, and E. urophylla hybrid genotypes that were planted on plantations at 13 sites and one experimental nursery. A total of 773 isolates of Calonectria were obtained from 683 plantation trees and nursery seedlings. Fifty-five representative isolates from all the surveyed sites and Eucalyptus species/genotypes were selected for molecular identification. These 55 isolates were identified by DNA sequence analyses based on the calmodulin (cmdA), histone H3 (his3), translation elongation factor 1-alpha (tef1), and ß-tubulin (tub2) gene regions, as well as a combination of morphological characteristics. The results indicated that these 55 isolates present one single species, Calonectria pentaseptata. Determined by sequences of cmdA, his3, tef1, and tub2 gene regions, only two genotypes were identified among the 55 representative isolates; 54 of these isolates share the same genotype, suggesting that the genetic diversity of Ca. pentaseptata collected during this study was relatively low. A growth study indicated that Ca. pentaseptata is a high-temperature species. The mating test results suggested that Ca. pentaseptata is heterothallic or lacks the ability to recombine to produce fertile progeny. Inoculation results showed that Ca. pentaseptata causes leaf blight and stem rot, resulting in tree death of the two widely planted Eucalyptus genotypes in southern China, and that the two genotypes differ significantly in their susceptibility to infection by Ca. pentaseptata. A selection program to develop Eucalyptus planting stocks with high levels of resistance to Calonectria leaf blight in China during the long-term should be urgently initiated.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Draft genome sequence data of Cercospora kikuchii, a causal agent of Cercospora leaf blight and purple seed stain of soybeans.

Cercospora kikuchii (Tak. Matsumoto & Tomoy.) M.W. Gardner 1927 is an ascomycete fungal pathogen that causes Cercospora leaf blight and purple seed stain on soybean. Here, we report the first draft genome sequence and assembly of this pathogen. The C. kikuchii strain ARG_18_001 was isolated from soybean purple seed collected from San Pedro, Buenos Aires, Argentina, during the 2018 harvest. The genome was sequenced using a 2 × 150 bp paired-end method by Illumina NovaSeq 6000. The C. kikuchii protein-coding genes were predicted using FunGAP (Fungal Genome Annotation Pipeline). The draft genome assembly was 33.1 Mb in size with a GC-content of 53%. The gene prediction resulted in 14,856 gene models/14,721 protein coding genes. Genomic data of C. kikuchii presented here will be a useful resource for future studies of this pathosystem. The data can be accessed at GenBank under the accession number VTAY00000000 https://www.ncbi.nlm.nih.gov/nuccore/VTAY00000000.

Diferenciação de bactérias do gênero Pseudomonas patogênicas ao cafeeiro por técnicas serológicas / Differentiation of bacteria of the genus Pseudomonas pathogenic to coffee by serological techniques

Há várias bactérias que causam problemas para o cafeeiro, incluindo Pseudomonas cichorii, P. syringae pv. garcae, P. syringae pv. tabaci, Burkholderia andropogonis e Xylella fastidiosa, todas elas já descritas no Brasil. Tentativas de diferenciar essas bactérias por testes serológicos de dupla difusão em ágar (dda), com antissoros produzidos contra células íntegras de P. s. pv. garcae, mostraram reações cruzadas, principalmente entre P. s. pv. garcae e P. s. pv. tabaci. Dessa forma, foram produzidos antissoros contra P. s. pv. garcae (linhagem patotipo IBSBF-248 - Coleção de Culturas de Fitobactérias do Instituto Biológico - IBSBF), obtidos por meio de imunizações de coelhos com antígenos de proteínas do complexo proteico da membrana (CPM). Esses antissoros foram testados por dupla difusão em agarose (dda) contra diversas formas de antígenos extraídos de P. cichorii, P. s. pv. garcae e P. s. pv. tabaci [células autoclavadas, células tratadas com formol, exopolissacarídeos (EPS), glicoproteínas (GP) da cápsula bacteriana, proteínas de membrana e suspensão bacteriana (SB) em NaCl 0,85%]. Os resultados mostraram que, dependendo do antígeno e do meio suporte da dupla difusão (com ou sem MgCl2 e/ou azul de tripano), os antissoros reagem somente com P. s. pv. garcae. Desse modo, esses antígenos podem ser usados para a rápida diagnose da mancha aureolada do cafeeiro nos testes de dda.

Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 - Phytobacteria Culture Collection of Instituto Biológico - IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test.

Diferenciação de bactérias do gênero Pseudomonas patogênicas ao cafeeiro por técnicas serológicas / Differentiation of bacteria of the genus Pseudomonas pathogenic to coffee by serological techniques

Há várias bactérias que causam problemas para o cafeeiro, incluindo Pseudomonas cichorii, P. syringae pv. garcae, P. syringae pv. tabaci, Burkholderia andropogonis e Xylella fastidiosa, todas elas já descritas no Brasil. Tentativas de diferenciar essas bactérias por testes serológicos de dupla difusão em ágar (dda), com antissoros produzidos contra células íntegras de P. s. pv. garcae, mostraram reações cruzadas, principalmente entre P. s. pv. garcae e P. s. pv. tabaci. Dessa forma, foram produzidos antissoros contra P. s. pv. garcae (linhagem patotipo IBSBF-248 - Coleção de Culturas de Fitobactérias do Instituto Biológico - IBSBF), obtidos por meio de imunizações de coelhos com antígenos de proteínas do complexo proteico da membrana (CPM). Esses antissoros foram testados por dupla difusão em agarose (dda) contra diversas formas de antígenos extraídos de P. cichorii, P. s. pv. garcae e P. s. pv. tabaci [células autoclavadas, células tratadas com formol, exopolissacarídeos (EPS), glicoproteínas (GP) da cápsula bacteriana, proteínas de membrana e suspensão bacteriana (SB) em NaCl 0,85%]. Os resultados mostraram que, dependendo do antígeno e do meio suporte da dupla difusão (com ou sem MgCl2 e/ou azul de tripano), os antissoros reagem somente com P. s. pv. garcae. Desse modo, esses antígenos podem ser usados para a rápida diagnose da mancha aureolada do cafeeiro nos testes de dda.(AU)

Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 - Phytobacteria Culture Collection of Instituto Biológico - IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test.(AU)

Diferenciação de bactérias do gênero Pseudomonas patogênicas ao cafeeiro por técnicas serológicas / Differentiation of bacteria of the genus Pseudomonas pathogenic to coffee by serological techniques

Há várias bactérias que causam problemas para o cafeeiro, incluindo Pseudomonas cichorii, P. syringae pv. garcae, P. syringae pv. tabaci, Burkholderia andropogonis e Xylella fastidiosa, todas elas já descritas no Brasil. Tentativas de diferenciar essas bactérias por testes serológicos de dupla difusão em ágar (dda), com antissoros produzidos contra células íntegras de P. s. pv. garcae, mostraram reações cruzadas, principalmente entre P. s. pv. garcae e P. s. pv. tabaci. Dessa forma, foram produzidos antissoros contra P. s. pv. garcae (linhagem patotipo IBSBF-248 - Coleção de Culturas de Fitobactérias do Instituto Biológico - IBSBF), obtidos por meio de imunizações de coelhos com antígenos de proteínas do complexo proteico da membrana (CPM). Esses antissoros foram testados por dupla difusão em agarose (dda) contra diversas formas de antígenos extraídos de P. cichorii, P. s. pv. garcae e P. s. pv. tabaci [células autoclavadas, células tratadas com formol, exopolissacarídeos (EPS), glicoproteínas (GP) da cápsula bacteriana, proteínas de membrana e suspensão bacteriana (SB) em NaCl 0,85%]. Os resultados mostraram que, dependendo do antígeno e do meio suporte da dupla difusão (com ou sem MgCl2 e/ou azul de tripano), os antissoros reagem somente com P. s. pv. garcae. Desse modo, esses antígenos podem ser usados para a rápida diagnose da mancha aureolada do cafeeiro nos testes de dda.(AU)

Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 - Phytobacteria Culture Collection of Instituto Biológico - IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test.(AU)

Diversity and potential impact of Calonectria species in Eucalyptus plantations in Brazil.

Species in the genus Calonectria (Hypocreales) represent an important group of plant pathogenic fungi that cause serious losses to plant crops in tropical and subtropical climates. Calonectria leaf blight is currently one of the main impediments to Eucalyptus cultivation in Brazil, and various species of Calonectria have been associated with this disease. Since most previous identifications were solely based on morphological characters, much of the published literature needs to be re-evaluated. The aim of this study was thus to identify and determine the phylogenetic relationships among species that occur in the Eucalyptus growing regions of Brazil by using partial sequences of the ß-tubulin, calmodulin, translation elongation factor 1-α and histone H3 gene regions. Based on extensive collections from soil and infected eucalypt leaf samples from plantations, phylogenetic inference revealed the Ca. pteridis complex to be the most common species complex present in Eucalyptus plantations in Brazil. By elucidating taxa in the Ca. pteridis, Ca. cylindrospora and Ca. candelabra species complexes, 20 novel Calonectria species were identified, and a new name in Calonectria provided for Cylindrocladium macrosporum as Ca. pseudopteridis.

Variación temporal a Microcyclus ulei en los clones de caucho FX 3864 y FX 4098 en condiciones controladas / Temporal variation to Microcyclus ulei in rubber tree clones FX 3864 and FX 4098 under controlled conditions

Este estudio tuvo como objetivo evaluar la variación temporal de los principales componentes fisiológicos asociados a la reacción de dos clones (FX 3864 y FX 4098) de Hevea brasiliensis a Microcyclus ulei en condiciones controladas. Las inoculaciones se realizaron en cámara húmeda a 23 ºC, una humedad relativa superior al 90% y un fotoperíodo de 12 h. Con un modelo de medidas repetidas en el tiempo y mediante correlaciones se analizaron cuatro variables: severidad de ataque (SA), intensidad de esporulación conidial (IE), tamaño de lesión (TL) y frecuencia de infección (FI). M. ulei generó síntomas en los dos clones a los 4 días después de la inoculación. Los signos de la enfermedad (IE= 3) se manifestaron a los 8 días en el clon FX 3864 y a los 12 días en el clon FX 098. FX 3864 presentó la mayor susceptibilidad a M. ulei (mayor severidad y una esporulación alta, IE= 4). En general, se observó una influencia del tiempo en la reacción de cada clon para las variables SA, IE y TL. Altas correlaciones significativas se encontraron entre SA y las variables IE, TL y FI, excepto en el día 12 para IE. Este estudio permite concluir que en condiciones controladas la intensidad y la frecuencia de los síntomas así como el grado de esporulación producidos por M. ulei cambian significativamente a través del tiempo y esta variación está influenciada a su vez por el nivel de susceptibilidad a M. ulei presentado por cada clon de H. brasiliensis.

This study aimed to evaluate the effect of temporal variation on the main physiological components associated with the reaction to Microcyclus ulei of two rubber tree clones of Hevea brasiliensis under controlled conditions. Inoculations were performed in a moist chamber at 23 °C, a relative humidity higher than 90% and a photoperiod of 12 h. The variables severity of attack (SA), conidial sporulation intensity (IE), lesion size (TL) and infection frequency (IF) were analyzed with a repeated over time measurements model and also with correlations. M. ulei produced symptoms (SA, TL, FI) in both rubber tree clones 4 days after inoculation. Signs of the disease (IE = 3) appeared in the FX 3864 at day 8th and in the FX 4098 at day 12th. FX 3864 presented higher susceptibility to M. ulei (greater severity and high sporulation, with IE = 4). In general, the time influenced the reaction of each clone, especially in the variables SA, IE and TL. High significant correlations were found between SA and the variables IE, TL and FI, except on day 12 for IE. It was concluded that intensity and frequency of symptoms and the degree of sporulation produced by M. ulei change significantly over time under controlled conditions and this variation is influenced also by the level of susceptibility to M. ulei showed by each clone of H. brasiliensis.

Controle de doenças foliares em trigo com equipamentos de pulverização de baixo volume de aplicação / Control of leaf diseases in wheat with low spray volume equipments

A utilização de equipamentos de pulverização com menor volume de aplicação no controle de doenças é uma tendência na agricultura. Portanto, este trabalho tem por objetivo determinar a eficiência de equipamentos com baixo volume de aplicação no controle de doenças foliares do trigo. O experimento foi conduzido em duas safras agrícolas e três cultivares no município de Santa Maria, RS. Os tratamentos constituíram-se de diferentes equipamentos de pulverização, sendo eles: pontas hidráulicas do tipo leque (XR 110015) e cone vazio sem indução de ar (TX-VK6), ambos com volume de aplicação de 120L ha-1, atomizadores rotativos de discos, utilizando baixo volume oleoso (BVO(r)), com volumes de aplicação de 24L ha-1 e 34L ha-1 e uma testemunha sem aplicação. As gotas geradas pelos equipamentos (densidade, diâmetro mediano volumétrico) foram avaliadas através da utilização de papéis hidrosensíveis. Também foram avaliadas a severidade da mancha amarela e da ferrugem da folha. A utilização de bicos hidráulicos com volume de aplicação de 120L ha-1 apresentaram as maiores eficiências no controle das doenças. O atomizador rotativo de discos com volume de aplicação de 34L ha-1 apresentou eficiência no controle das doenças semelhantes ao bico hidráulico. A densidade de gotas foi superior a 40 gotas cm-2 em todos os tratamentos, mesmo nos tratamentos com atomizadores rotativos de disco com baixo volume de aplicação. O controle das doenças não apresentou relação direta com a cobertura mensurada pelos cartões hidrosensíveis.(AU)

The use of spraying equipments with lower spray volume at disease control is a tendency in agriculture. The study aimed to determinate the efficiency of low spray volume at the wheat leaf diseases control. The experiment was carried during two harvest years and three cultivars in Santa Maria, RS. The treatments were constituted of different spraying equipments: hydraulic nozzle (XR 110015) and hollow cone (TX-VK6), both with spray volume of 120L ha-1, rotary-disk atomizer using low volume in oil, with spray volume of 24L ha-1 and 34L ha-1 and a control treatment without application. The droplets generated by the equipments (density, medium volumetric diameter) were evaluated throughout the use of water sensible cards. Also were evaluated the yellow leaf blight and leaf rust. The use of hydraulic nozzle at spray volume of 120L ha-1 showed higher efficiency on the disease control. The rotary-disk atomizer with spray volume of 34L ha-1 showed similar efficiency in the disease control of hydraulic nozzle. The droplet density was superior to 40 droplets cm-2 in all treatments, also at the rotary-disk treatments with low pulverization rates. The disease control did not show direct relation with the coverage measured by the water sensible cards.(AU)