Mexican Journal
of Phytopathology

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Epidemiological etiology of Erysiphe sp. and putative viral and phytoplasma-like symptoms in Ayocote bean (Phaseolus coccineus)

By María José Armenta Rojas, Norma Ávila Alistac, María del Carmen Zúñiga Romano, Gerardo Acevedo Sánchez, Alfonso Muñoz Alcalá, Rene Gómez Mercado, Juan José Coria Contreras, Diana Gutiérrez Esquivel, Serafín Cruz Izquierdo, Ivonne García González, Oscar Bibiano Nava, Gustavo Mora Aguilera*

* Corresponding Author. Email: / Institution: Colegio de Postgraduados

Accepted: 25/December/2024 – Published: 12/February/2024 – DOI: https://doi.org/10.18781/R.MEX.FIT.2310-7

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Abstract Introduction/Objective. Ayocote bean (Phaseolus coccineus) has potential as a source of resistance in breeding programs because it exhibits greater tolerance to plant pathogens than P. vulgaris. However, its sanitary characterization is insipient; therefore, the purpose of this work was to carry out an etiological-epidemiological diagnosis, with emphasis on presumptive symptoms of viral and phytoplasmic organisms, and a typical fungal signs of powdery mildew.

Materials and Methods. A plot (50 x 62 m) of flowering Ayocote bean was selected. It was divided into 80 (8 x 10) quadrats (6 x 6 m) and 720 subquadrats (2 x 2 m). From 25 plants with powdery-mildew-type leaf symptoms, mycelium was collected with adhesive tape for light microscopy observation and taxonomic identification. Length-width measurements were made on 60 conidia. Pure mycelium collected in situ and ex situ from 1-5 leaflets/plant was used for genomic analysis by PCR with universal primers ITS1 and ITS4. Samples were sequenced in Macrogen Inc. Korea. A total of 63 plants and 121 trifoliate leaves with viral and phytoplasmic symptoms were collected by direct sampling. In 88/121 samples, genomic analysis was performed by PCR with universal primers for Potyvirus (1), Begomovirus (2), and Phytoplasmas (1). Sequence editing and analysis were performed in SeqAssem and BLASTn/GenBank. Phylogenetic constructions were developed in Mega 11 with MUSCLE, Maximum Likelihood (ML), and HKY substitution model (1000-Bootstrap). Putative powdery mildew severity (%), flower damage (%), Macrodactylus sp. adult density, and plant vigor (%) were evaluated in 80 quadrats (3subquadrats/quadrat) with App-Monitor®v1.1 configured with a 5-class scale. In GoldenSurfer® v10, Kriging geostatistical analysis was performed to determine the spatial interrelationship between these variables.

Results. Erysiphe vignae was identified as associated with powdery mildew of P. coccineus. The fungus, with hyaline, ovoid to ellipsoid conidia measuring 31.74 ± 0.3419 μm x 15.11 ± 0.1579 μm, without the presence of fibrosin bodies, had 100% genomic homology. This is the first report in Mexico. With average July-August temperature and relative humidity of 16.3 °C (±5.8) and 92.8 % (±10.7), respectively, powdery mildew leaf incidence and severity were 65.3 and 22.7 % (±16.9, range: 0 - 66.5 %), respectively. The most inductive focus (60- 80 % severity) had an aggregate e 4-quadrat pattern (96 m2, lag = 4 and σ2-s = 450). Inoculum dispersal was significantly associated with dominant North-South winds and plant vigor (lag = 4 and σ2-s = 470). Flower damage was inconclusive in its spatial association with powdery mildew and Macrodactylus sp. suggesting uncorrelated events. No Potyvirus, Begomovirus, or Phytoplasmas were detected associated with yellowing, leaf distortion, mosaic, internode shortening, and other symptoms observed in situ. This confirms the relative tolerance/resistance reported for P. coccineus.

Conclusion. E. vignae (Erysiphales: Erysiphaceae) associated with P. coccineus is reported for the first time in Mexico with moderate to intense epidemic level, which indicates its susceptible condition to this fungus. However, negative results for Potyvirus, Begomovirus, and Phytoplasmas, validate the apparent tolerance/ resistance of P. coccineus to these organisms.

Keywords: Erysiphe vignae, Macrodactylus sp. powdery mildew, Potyvirus, Begomovirus

Figura 2. Morphological and genomic identification of powdery mildew in Ayocote bean (Phaseolus coccineus). A. hyaline, ovoid to ellipsoid conidia; B-C. cylindrical and erect conidiophores; D-F. germinating conidia; G. Amplification of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (~500 bp) of five samples of cenicilla DNA (1-5), two positive PCR controls (+) belonging to the ITS region of Alternaria and Fusarium genera, 1 kb molecular weight marker (M) plus Invitrogen and negative PCR control (-); H. phylogenetic tree performed by Maximum Likelihood (ML) and the Hasegawa-Kishino-Yano substitution model with 1000 Bootstrap replications, based on ITS region of fungal sequences belonging to Erysiphe genus (Table 2). The study sequences are: FAC6, FAC7, FAC8 and FAC9 (red dot). Oidium sp. (accession number: EU377475) was included as outgroup

Figura 3. Presumptive field symptoms of phytoplasma infection in Ayocote bean (Phaseolus coccineus). A. Putative symptoms to phytoplasmas. Including leaf deformation, brown or violet coloration, obvious purplish coloration on veins of back young leaves, stunting, slight yellowing of leaves; B. Examples of six trifoliate leaves showing presumptive symptoms of phytoplasmas, included in PCR diagnosis; C. Agarose gel electrophoresis of 24 samples processed by nested PCR. PCR-amplified positive controls only.

Figura 4. Geostatistical Kriging contour maps and variograms of phytosanitary variables and vigor in Ayocote bean. A. Powdery mildew severity, B. Flower damage, C. Density of Macrodactylus sp. adults and D. Plant canopy. For adult analysis, cumulative of three subquadrants per quadrant was calculated. For the analysis of powdery mildew severity, flower damage, and plant canopy, the maximum damage obtained per quadrant was calculated. Omnidirectional variograms were obtained by the Spherical method. X-axis = distance-lag in quadrants and Y-axis = variance (σ2)

Figure 1. Sampling methodology to identify and assess severity of fungal, presumtive viral/phytoplasmal symptoms, and entomological signs on Phaseolus coccineus. A. 13mpx image at 50m using DJI® Phantom-3 drone, showing quadrantization of experimental plot. Yellow-lines correspond to 6x6 m quadrants, and white-lines to 2x2 m subquadrants. Asterisks indicate randomly selected subquadrants/quadrants; B. Field quadrant marking with wooden-stakes and a slat-net; C. Selection of subquadrant by placing wooden frame 1x1 m for assessment guidance; D. Dotted mosaic symptoms (left) and vein clearing (right), putative to virosis. Plants marked with stakes for traceability samples; E. Generalized yellowing with growth reduction (left), mosaic with leaf deformation (right) presumptive viral; F. Leaf symptom with white fungal mycelial growth putative to powdery mildew; G. Front leaflet showing white mycelial growth. H. Macrodactylus sp. adults, and flowering color morphology of P. coccineus. Note some petals showing small white-spots (see arrows).

Table 1. Primers, sequences, and amplicon size for genomic identification of Potyvirus, Begomovirus, Phytoplasmas and eukaryotic microorganisms in P. coccineus plants exhibiting signs of powdery mildew and putative virus and phytoplasma symptoms.

Table 2. Sequences obtained from the NCBI Genebank used to construct the phylogenetic tree for comparison with amplicon sequences obtained from four samples of powdery mildew fungus present in P. coccineus plants.