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Cultivation of yellow dragon fruit (Selenicereus megalanthus) in Peru has recently expanded (Verona-Ruiz et al. 2020). In August 2021, approximately 170 of 1,110 dragon fruit cuttings (15.3%) in the university's nursery (6°26'10'' S; 77°31'25'' W) showed basal rot symptoms. Initial symptoms included small brown spots on the base of stems, expanding towards the top that became soft and watery. All symptomatic plants eventually died, i.e., a severity of 100%. The disease was more prevalent on cuttings during the rooting phase than on well-established cuttings. We collected five symptomatic cuttings from throughout the nursery. Four sections of 1 × 1 cm2 of tissue adjacent to the diseased area were excised from each cutting, immersed for 1 min in 2% NaClO, rinsed twice with sterile distilled water, placed on potato dextrose agar (PDA) medium (four sections per Petri plate, five plates), and incubated at 25°C for 7 days. Morphologically similar mycelia grew from all sections, and five monosporic isolates were obtained, one per plate. Colonies grew fast, reaching 60 to 64 mm in 7 days, and produced violet-white cottony aerial mycelia with orange sporodochia on PDA, and abundant macro- and microconidia on synthetic nutrient-poor agar. Macroconidia were straight to slightly curved, typically with 2 to 3 septa, 16.6 to 23.3 × 1.7 to 3.7 µm (n = 30); microconidia were oval or kidney-shaped, and commonly hyaline, 6.7 to 16.4 × 2.5 to 4.7 µm (n = 40). Genomic DNA was extracted from isolate AFHP-100, then the ITS region and the TEF1 and RPB2 partial genes were amplified and sequenced (Accession numbers PP977433, OR437358, PP537149) following Gardes and Bruns (1993) and O'Donnell et al. (1998). We conducted a BLASTn search of ITS sequence against the NCBI "nr" database and local 'megablast' searches of TEF1 and RPB2 sequences against FUSARIUM-ID v.3.0 (Torres-Cruz et al. 2022). We found 100%, 98.19 to 99.84%, and 98.81 to 99.76% identities in ITS, TEF1, and RPB2 sequences, respectively, to the ex-epitype and other reference strains of Fusarium oxysporum (CBS 144134, NRRL26406, among others). A maximum likelihood phylogenetic analysis with a TEF1-RPB2 concatenated dataset with FUSARIUM-ID sequences also showed isolate AFHP-100 was F. oxysporum. A pathogenicity test was carried out by inoculating wounded healthy roots of three cuttings with submersion in a 5 × 106 conidia/ml suspension for 25 min. Then, the inoculated plants were planted in sterile soil. One cutting with wounded roots submerged in sterile water served as a control. In parallel, sterile soil was inoculated with 20 mL of the conidial suspension, and another three healthy cuttings were planted. A cutting planted in noninoculated soil also served as a control. Basal rot symptoms developed in all inoculated plants after 25 days. After re-isolation, the same fungus, corroborated based on micromorphology and TEF1 sequence (PP335689), was recovered, fulfilling Koch's postulates. The isolate was deposited in the KUELAP Herbarium (voucher KUELAP-3214), located and administered by the National University Toribio Rodriguez de Mendoza de Amazonas, in Chachapoyas, Peru. Fusarium oxysporum has been reported to cause basal stem rot in Bangladesh and Argentina (Mahmud et al. 2021; Wright et al. 2007), and stem blight in Malaysia (Mohd Hafifi et al. 2019) on dragon fruit. This is the first report of F. oxysporum causing basal rot in S. megalanthus in Peru. This fungus is among the most destructive plant pathogens, and the rapid expansion of the crop in Peru requires a comprehensive knowledge of the biotic factors influencing production. Therefore, this report is foundational to implementing proper control strategies.
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The Peruvian Amazonian native cacao faces ongoing challenges that significantly undermine its productivity. Among them, frosty pod rot disease and cadmium accumulation result in losses that need for effective and environmentally safe strategies, such as those based on bacteria. To explore the biological resources in the cacao soil, a descriptive study was conducted to assess the diversity of culturable bacteria across three production districts in the Amazonas region: La Peca, Imaza, and Cajaruro. The study also focused on the functional properties of these bacteria, particularly those related to the major issues limiting cacao cultivation. For this purpose, 90 native bacterial isolates were obtained from the cacao rhizosphere. According to diversity analysis, the community was composed of 19 bacterial genera, with a dominance of the Bacillaceae family and variable distribution among the districts. This variability was statistically supported by the PCoA plots and is related to the pH of the soil environment. The functional assessment revealed that 56.8% of the isolates showed an antagonism index greater than 75% after 7 days of confrontation. After 15 days of confrontation with Moniliophthora roreri, 68.2% of the bacterial population demonstrated this attribute. This capability was primarily exhibited by Bacillus strains. On the other hand, only 4.5% were capable of removing cadmium, highlighting the biocontrol potential of the bacterial community. In addition, some isolates produced siderophores (13.63%), solubilized phosphate (20.45%), and solubilized zinc (4.5%). Interestingly, these traits showed an uneven distribution, which correlated with the divergence found by the beta diversity. Our results revealed a diverse bacterial community inhabiting the Amazonian cacao rhizosphere, showcasing crucial functional properties related to the biocontrol of M. roreri. The information generated serves as a significant resource for the development of further biotechnological tools that can be applied to native Amazonian cacao.
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The thread blight disease (TBD) of cacao ( Theobroma cacao) in the department of Amazonas, Peru was recently reported to be caused by Marasmius tenuissimus (sect. Neosessiles). This same species is known to be the main causal agent of TBD in West Africa. However, some morphological characteristics, such as the presence of rhizomorphs, the almost exclusively white color, and pileus sizes less than 5 mm, among others, differ to the description of M. tenuissimus. Therefore, we aimed to conduct a taxonomic revision of the cacao-TBD causal agent in Peru, by using thorough micro and macro morphological, phylogenetic, and nuclear and mitochondrial genomic approaches. We showed that the causal agent of TBD of cacao in Amazonas, Peru, belongs to a new species, Marasmius infestans sp. nov. This study enriches our knowledge of species in the sect. Neosessiles, and strongly suggests that the M. tenuissimus species complex is highly diverse.
Assuntos
Cacau , Filogenia , Doenças das Plantas , Cacau/microbiologia , Cacau/genética , Doenças das Plantas/microbiologia , Peru , GenômicaRESUMO
Mangoes (Mangifera indica L.) are one of the most important export fruits in Peru and anthracnose, caused by several species in the Colletotrichum gloeosporioides species complex (CGSC), is one of their main postharvest diseases (Alvarez et al. 2020). Balsas is the major mango producing district in the Amazonas department, where farmers practice intercropping in orchards mostly of less than 5 ha (Cabezudo Cerpa 2022). In July 2021, mango fruits cv. Kent with anthracnose were detected at an incidence of 55 to 80% during postharvest in Balsas. Symptoms included sunken dark brown lesions with appearance of orange conidiomata at advanced stages of the disease. We collected two samples of infected mangoes from a farm located at 6°51'01" S, 77°59'48" W (1088 m.a.s.l.). One axenic culture (INDES-AM1) was obtained from a hyphal tip of a monosporic colony and cultivated on PDA medium at 25 °C in the dark. The growing rate of the colony was 8.1 mm.day-1. Conidia were hyaline, guttulate, unicellular and cylindrical with narrowing center, with dimensions of 15.8 to 23.5 × 4.5 to 7.6 µm (mean = 18.6 ± 0.03 × 6.0 ± 0.02 µm, SE, n = 50), consistent to the CGSC (Weir et al. 2012). Appressoria were dark brown, and ovoid to slightly irregular in shape, ranging from 5.3 to 10.1 × 4.7 to 8.3 µm (mean = 7.9 ± 0.02 × 6.0 ± 0.02 µm, SE, n = 50). Koch's postulates were fulfilled on mature mango fruits of the same cultivar and from the same district. Mangoes were washed with detergent and left to dry before inoculation. PDA-mycelial plugs of 0.5 cm wide were transferred on two different locations of two fruits, with four replicates. One location was previously wounded with five needle punctures of 3 mm depth. The inoculated fruits were maintained in a moist chamber at ambient light and temperature (18.9 ± 0.5 °C, SE). Symptoms appeared three-to-five days post inoculation (dpi), and the superficial diameter of the lesions were 8.3 ± 1.5 and 3.6 ± 2 mm with the invasive and the superficial inoculation approaches, respectively, at five dpi. Lesions were very similar to original symptoms. Macro and micromorphological characteristics of the re-isolated fungal colonies were the same as isolate INDES-AM1. Molecular identification of the pathogen was carried out following Weir et al. (2012). Total DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, Wisconsin) and the ribosomal internal transcribed spacer (ITS), and partial sequences of the chitin synthase (CHS1), actin (ACT), ß-tubulin 2 (TUB2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear genes were sequenced (Accession numbers: OP425395, OP440444, OP440442, OP440443, OP555062, OP555063). ITS, CHS1, ACT, TUB2, CAL, and GAPDH sequences were 98.6, 100, 100, 99.5, 100, and 99.08% identical to Colletotrichum asianum type strain ICMP 18580 sequences, respectively. Additionally, a bootstrapped maximum likelihood midpoint-rooted phylogeny with a multilocus dataset with the six sequences from reference strains of C. asianum and closely related species within the CGSC revealed that strain INDES-AM1 is C. asianum. This species has been found causing anthracnose on M. indica in at least 15 different countries in Africa, America, Asia, and Oceania (Weir et al. 2012). It was originally described from coffee and has multiple other hosts (Prihastuti et al. 2009; Lima et al. 2015). To the best of our knowledge, this is the first report of C. asianum infecting mangoes in Peru.
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Peru is the second largest producer of organic cocoa and one of the most important suppliers of fine aroma cocoa beans in the world (Sánchez et al. 2019). The fine aroma cocoa produced by smallholder farmers in the Bagua and Utcubamba Provinces, Amazonas Department, under the name of "Cacao Amazonas Peru", is protected by the Peruvian appellation rules (Díaz-Valderrama et al. 2020). Despite this importance, native diseases of the crop (Theobroma cacao) are poorly documented due to difficulty of access in this region. In November 2020 we conducted expeditions into Imaza District (4°47'09.4"S 78°16'51.6"W), a significant producer of fine aroma cocoa in terms of number of cultivated plots (4,651 out of 6,505 total in the Bagua Province) (INEI 2012). We visited 20 farms of < 2-ha in size; in 19 of these small farms, T. cacao trees were found infected with a white fungal thread blight and rhizomorphs covering branches and leaves. Disease incidence ranged from 90 to nearly 100%, and severity exceeded 80% on the eight farms with the most deficient phytosanitary management. Heavily infected leaves were hanging on branches by mycelial threads, harboring tiny (0.5 to 5.3 mm broad) white mushrooms. These symptoms and signs correspond to the thread blight disease constellation (TBD) of cacao caused by various species of Marasmius and Marasmiellus (Amoako-Attah et al. 2020). Mushrooms lacked a collarium, and their stipes were absent or rudimentary (< 2-mm long) and eccentric, consistent with Marasmius tenuissimus (Tan et al. 2009). Axenic cultures were obtained by surface sterilization of mycelium threads with 2% NaClO, rinsed three times in sterile water, plated on potato dextrose agar medium (PDA), and incubated for 7 days at 25°C. Hyphae was non-pigmented with clamp connections, consistent with the genus Marasmius. We extracted the DNA of isolate INDES-AFHP31 using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin) and sequenced the rDNA internal transcribed spacer 1 and 2 intervening the 5.8S subunit (ITS), and the 28S subunit (LSU) (Accession numbers: OM720123 and OM720135) according to Aime and Phillips-Mora (2005). The ITS and LSU sequences were 97.92 to 98.79% and 99.07 to 99.30% identical, respectively, with published sequences from M. tenuissimus from Ghana (Amoako-Attah et al. 2020). The pathogenicity test was conducted by inoculation of ten healthy cacao leaves with 7-day-old mycelium PDA discs of isolate INDES-AFHP31. An equal number of healthy cacao leaves were inoculated with PDA discs without mycelium as control. The observation of TBD symptoms and signs in the non-control set of cacao leaves starting at 3 days post inoculation, and the re-isolation of the same fungus from infected tissue confirmed its pathogenicity on cacao. Isolate INDES-AFHP31 was deposited as a dried culture into the herbarium Kuélap of the Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (voucher KUELAP-2251). Marasmius tenuissimus was originally reported from dead and living twigs and leaves of unidentified dicotyledonous trees from Indonesia, Brazil, and Bolivia (Singer 1976). However, it was first associated with TBD of cacao in Ghana in 2020, being the most frequently TBD-causing fungus isolated in the country (Amoako-Attah et al. 2020). Its discovery in 19 of the 20 surveyed cacao farms in Imaza District, Amazonas, Peru, reveals its importance as a cacao pathogen in the Western hemisphere.
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Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the ß-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.
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Alfalfa (Medicago sativa) is the most cultivated fodder crop in Peru with 172,000 ha cultivated (MINAM 2019), and Arequipa is the top producing region with 40% of the national production in 2015 (Santamaría et al. 2016). In January-April 2019 (av. 20°C and 70% RH), most alfalfa fields in Majes-Pedregal, Arequipa were affected by an unidentified foliar disease. One of the fields was located at the farm of the Universidad Nacional de San Agustín de Arequipa (16°19'29.6" S, 72°12'59.9" W). Symptoms appeared as elliptical light brown spots witdark brown borders (Fig. S1a and b). The field (~60 × 60 m) was divided into ~30 × 12 m sections and two plants in each section were collected (20 plants total). Plants were digitized and the leaflet diseased area was calculated with ImageJ 1.53a, from which an incidence of 100% and a severity of 38.7 ± 4.4 % were estimated. Microscopical observations at the leaflet spots revealed consistently the presence of oblong multiseptated conidia (23.6-42.8 × 16.5-25.2 µm; av. 33.3 × 20.9 µm; n = 40) of the genus Stemphylium (Simmons 1969; Woudenberg et al. 2017) (Fig. S1c). We obtained 10 pure cultures by placing conidia from the spots directly onto potato dextrose agar medium with the aid of stereoscope and sterile forceps. Two isolates (UNSA-StemV01 and UNSA-StemV02) were incubated further until ascospore production at room temperature with no special light stimulus. After 45 days of growth, globose pseudothecia and ellipsoidal ascospores (25.4-38.7 × 11.2-16.6 µm; av. 31.9 × 13.7 µm; n = 30) formation occurred (Fig. S1d and e). We extracted the DNA from these two isolates using Wizard® Purification Kit (Promega Corp., Madison, WI) and sequenced the internal transcribed spacer 1 and 2 intervening 5.8S rDNA subunit (GenBank accessions: MT371236-37), and the glyceraldehyde-3-phosphate dehydrogenase (MT375513-14) and the calmodulin (MT375515-16) genes, highly resolutive markers to identify Stemphylium species, following Woudenberg et al. (2017). We retrieved sequence data available from 43 isolates of nine Stemphylium species (Han et al. 2019; Woudenberg et al. 2017), and built a mid-point rooted phylogeny with the three-loci concatenated data set (Fig. S2). We identified our isolates as S. vesicarium (Fig. S2). Koch's postulates were fulfilled by spray-inoculation with conidia from isolate UNSA-StemV01 suspended in sterile water (1×104 / mL) to two healthy 50-day old alfalfa plants growing on pots in the university greenhouse (av. 25°C and 70% RH). Two plants sprayed with sterile water without conidia served as control. Symptoms appeared after 21 days of inoculation, and when conidia were re-isolated, they were the same as originally obtained. No symptoms developed in the control plants. This confirmed that S. vesicarium is the causal agent of the alfalfa disease in Majes-Pedregal, identified as Stemphylium leaf spot. revious studies documented S. vesicarium on asparagus and onion in Peru (Castillo Valiente 2018; Vásquez Salas 2018; Vásquez Sangay 2013), but molecular characterization has only been applied to S. lycopersici from potatoes (Woudenberg et al. 2017). Stemphylium vesicarium has been documented in various crops, including alfalfa, and countries in Europe, North America, Africa, Asia and in Australia and New Zealand (Han et al. 2019; Woudenberg et al. 2017). This occurrence is the first report of S. vesicarium on alfalfa in Peru. The disease compromises the quality of this fodder crop, so actions need to be taken in Arequipa.