ABSTRACT
Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.
ABSTRACT
The guava (Psidium guajava L.) is a plant native to the tropical region of America. In Mexico, the area established with guava cultivation is 20,525 ha (SIAP 2021). Guava is commonly consumed as fresh fruit, being rich in nutrients such as vitamins and minerals (Murthy et al. 2020). During October 2020, in the municipality of Cocula (18.207835N, 99.670322W, 595 m above sea level), Guerrero, Mexico, severely infected immature guava fruits were observed. The incidence of disease in 150 sampled fruits was 12%. Were collected fifteen symptomatic fruits. The symptoms were circular to irregular dark brown spots that varied in size (0.5 to 2.5 cm). From symptomatic fruits, tissues were cut approximately 3 x 3 mm and disinfested with 1% NaOCl, washed three times with sterile distilled water, and transferred to PDA medium amended with streptomycin and tetracycline, and incubated at 28°C. Developing colonies were retransferred to new culture PDA medium, and purified by hyphal tip technique. Two representative isolates (PHYGUA7 and PHYGUA3) were selected for morphological and molecular characterization. After 15 days in PDA at 28°C in an incubator, colonies were flat, irregular, granular and greenish gray, pycnidia were black, granular, and grouped. The conidia were hyaline and ellipsoid, unicellular and smooth-walled, 7-11×5-6.5 µm (n=50), these characteristics were consistent with those described for the fungus Phyllosticta capitalensis (Wikee et al. 2013). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), the actin gene (ACT), and the translation elongation factor 1-alpha (EF-1α) gene, using primers ITS1 and ITS4, ACT-512F/ ACT-783R, and EF1-728F/EF1-986R, respectively (White et al. 1990; Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (PHYGUA7: OP810947, PHYGUA3: OP810948 for ITS, PHYGUA7: OP819845, PHYGUA3: OP819846 for ACT, and PHYGUA7: OP819847, PHYGUA3: OP819848 for EF-1α). Phylogenetic analysis using maximum likelihood concatenated sequences ITS, ACT and EF-1α with MEGA X, indicated that PHYGUA7 and PHYGUA3 isolated grouped with P. capitalensis (CPC 18848 type strain). For pathogenicity test of P. capitalensis, 15 healthy immature fruits in a field experiment in the fruits on the trees, and 15 healthy mature guava fruits (detached fruits) were superficially disinfected with 70% ethanol, wounded with a sterile toothpick, and inoculated at two equidistant points by inserting PHYGUA7 isolate mycelium. As a control treatment, 10 healthy immature fruits and 10 healthy mature fruits were only injured with a sterile toothpick. After 3 days symptoms were observed in mature fruits and numerous dark pycnidia developed, and seven days later symptoms were observed in immature fruits in all the points inoculated with the PHYGUA7 isolate, similar to the symptoms observed in the field. The control fruits remained asymptomatic. The fungus P. capitalensis was re-isolated from inoculated fruits, thus confirming Koch's postulates. In Mexico P. capitalensis has been reported in Mangifera indica, Epidendrum sp., and Schomburgkia tibicinis (Farr and Rossman, 2022). In Egypt and China P. capitalensis causes black spot on guava fruits (Arafat 2016; Liao et al. 2020). To our knowledge, this is the first report of P. capitalensis as the cause of brown spot on immature guava fruit in Mexico. This research provides relevant information to the design of disease management strategies.
ABSTRACT
The agave crop (Agave angustifolia), is of economic importance for Mexico, for the agave is made mainly an alcoholic beverage called locally mezcal. In the state of Guerrero, in the municipality of Huitzuco de los Figueroa (18.2510026N, 99.2320182W, 1196 m above sea level), a severe disease affecting agave leaves was detected. The field symptoms consisted of pale to brown dark descending lesions, covering >50% of the leaf surface, in which the presence of pycnidia was observed. In an estimated area of 0.5 ha, the estimated incidence was 67% (n=100 plants). Symptomatic fragments from leaves (approximately 0.5 cm) were taken, superficially disinfected with 1% NaClO, and rinsed twice with sterile distilled water. Then they were transferred to potato dextrose agar (PDA) medium, and incubated at 28 °C. After five days, twelve representative isolates were selected and purified by the hyphal tip technique. In the PDA medium, the colonies were initially light gray, later they became dark, and after 22 days of incubation, the development of numerous dark pycnidia was observed on the surface of the medium. Initially, immature hyaline conidia, unicellular, oval, and double-walled were observed. The mature conidia were dark brown, oval, with one septum and longitudinal striation, and measured 17.5 to 27 [average 25.3 µm; n=50] × 10.5 to 15.7 [average 13.9 µm; n=50]. Based on the morphological characteristics, the fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). Isolates LAS3 and LAS4 were used for molecular identification, this was done by amplifying the regio internal transcribed spacer (ITS) of rDNA with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1-alpha ( EF-1α) genes using primers EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (LAS3; ON391564 and LAS4; ON391565 for ITS, and LAS3; ON368190 and LAS4; ON368191 for EF-1α). BLASTn analysis sequences of isolated LAS3 and LAS4 revealed for ITS 98.6% identity with L. theobromae (MK934699.1), and for EF-1α indicated 100% identity (MF422024.1). From concatenated sequences ITS-EF-1α regions, a phylogenetic analysis was carried out in MEGA X software, using the Maximum Likelihood and Kimura 2-parameter model with 1,000 bootstraps replicated; isolates LAS3 and LAS4 were clustered in the clade of the members of L. theobromae strains CAA006 (Alves et al. 2006), and INTA-IMC 1601 (Perez et al. 2018). The pathogenicity tests were carried out on 10 healthy 3 year-old agave plants, in which the mycelium of the LAS4 isolate was inserted at three equidistant points/leaf, using a sterile toothpick. Five healthy agave plants were inoculated only with sterile PDA as control treatment. The inoculated plants were covered with transparent plastic bags and housed in a greenhouse at 28 °C. After seven days, similar symptoms to those observed in the field were observed in all inoculated plants. Control plants did not develop symptoms. The fungus L. theobromae was re-isolated again from the infected leaves, fulfilling Koch's postulates. In China, L. theobromae has been reported as the cause of leaf rot on A. sisalana (Xie et al. 2016). To our knowledge, this is the first report of L. theobromae causing leaf rot on A. angustifolia in Mexico. This research is useful to design management strategies for leaf rot disease for local farmers of A. angustifolia.
ABSTRACT
The agave (Agave spp.) is an important crop in México, with 120,897 ha grown mainly for alcoholic beverage production (SIAP, 2019). In September 2020, in the municipality of Huitzuco de los Figueroa (18.328692 N; 99.3998 W), Guerrero State, México, a serious disease was observed affecting Agave angustifolia. Disease incidence was 8% of 150 plants sampled over an approximate area of 2.5 ha. Initial symptoms of soft rot of the bud developed and produced an abundant exudate which appeared from the apical part to the base of the plant. In severe infections, the plants showed total maceration of the bud, and consequently death of the plants was observed. Symptomatic plant tissue was superficially disinfected with 1% NaOCl for 30 s, and rinsed in sterile water three times. The disinfected tissues were macerated and with a loop spread in Nutrient Agar. The plates were incubated at 28 ° C for 2 days. Yellowish bacterial colonies were isolated, and eight colonies were selected for characterization. The bacterial strains were gram negative and rod-shaped, negative for fluorescent pigment tests and Kovacs' oxidase. Two isolates designated AGA1 and AGA2 were identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F / 1492R (Lane 1991), and partial fusA, rpoB, and gyrB genes (Delétoile et al. 2009). Sequences were deposited in GenBank, with the accession numbers for 16S rRNA, AGA1 as MW548406 and AGA2 as MW548407; for specific genes fusA (AGA1 = MW558445, AGA2 = MW558446), rpoB (AGA1 = MW558447, AGA2 = MW558448) and gyrB (AGA1 = MW558449, AGA2 = MW558450), and they were compared with the sequences available in GenBank using BLASTn. 16S rRNA gene sequences for AGA1 and AGA2 aligned with Pantoea dispersa (MT921704.1, 99.9% identity). Housekeeping genes also aligned 99 to 100% to P. dispersa (fusA = 100%, CP045216.1; rpoB = 99.8% MH015167.1 and gyrB = 99%, MK928270.1). Phylogenetic analysis of concatenated genes showed that strains AGA1 and AGA2 cluster with P. dispersa. To confirm pathogenicity, eight plants of six-month-old A. angustifolia were inoculated with strain AGA1 using sterile toothpicks dipped in 108 CFU/ml bacterial suspension. The toothpicks were inserted in the middle part of the bud. Four plants were inoculated with sterile water as control. The plants were covered with plastic bags and housed in a greenhouse (average temperature and relative humidity of 25 ° C and 85%, respectively). Pathogenicity tests were repeated two times. After seven days, all inoculated plants developed symptoms similar to those observed in the field. Control plants did not show symptoms. From the plants that showed symptoms, the pathogen was reisolated again and was identified by morphological and molecular characterization, following the method previously described, fulfilling Koch's postulates. In México, Erwinia cacticida and Pantoea ananatis has been previously reported on A. tequilana that as causing soft rot and red leaf ring, respectively (Jimenez-Hidalgo et al. 2004; Fucikovsky and Aranda 2006). To our knowledge, this is the first report of P. dispersa causing bud soft rot on A. angustifolia in México. More studies monitoring and control strategies of bud soft rot on A. angustifolia are required.
