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Agrociencia
versión On-line ISSN 2521-9766versión impresa ISSN 1405-3195
Agrociencia vol.51 no.2 Texcoco feb./mar. 2017
Plant Protection
Mycoflora associated to leaf spots and blights in wheat (Triticum aestivum L.) under irrigation conditions in El Bajío, Mexico
1Programas de Sanidad Forestal y Agrícola, Trigo y Biotecnología, Campo Experimental Bajío, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP-CEBAJ), Km. 6.5, Carretera Celaya-San Miguel de Allende, 38110. Celaya, Guanajuato.
2Departamento de Protección Vegetal, Universidad Autónoma Chapingo. 56230. Chapingo, Estado de México.
3Programa de Trigo y Avena, INIFAP, Campo Experimental Valle de México, Km. 13.5, Carretera Los Reyes-Texcoco, Coatlinchán, Texcoco, Estado de México, 56250.
The production of wheat (Triticum aestivum L.) under irrigation in the Bajio region, Mexico, is mainly affected by leaf rust (Puccinia triticina Eriks.) and stripe rust (Puccinia striiformis f. sp. tritici). In recent years, other symptoms were observed in this region, such as leaf spots and blights, which also limit the productivity of the wheat varieties. In order to identify the fungi associated with these symptoms, leaves with blights and leaf spots were collected from 13 municipalities in the States of Guanajuato, Michoacan and Jalisco, Mexico, during the fall-winter agricultural cycles of 2012-2013 and 2013-2014. The fungi identification was carried out using morphological methods, amplification and sequencing of internal transcribed spacer regions (ITS). The identified pathogenic fungi were: Bipolaris sorokiniana (Sacc.) Shoem. (Synonym: Helminthosporium sativum Pammel, CM King and Bakke), Alternaria alternata (Fr.:Fr), Septoria tritici, Fusarium proliferatum (Matsushima) Nirenberg, F. equiseti (Corda) Sacc., F. moniliforme (J. Sheld) (Syn. F. verticillioides (Sacc.) Nirenberg), Curvularia spp., Cladosporium spp., the saprophytes Nigrospora spp., Torula spp., Epicoccum spp., and the antagonist fungus Chaetomium globosum Kunze ex. Fr. Although the phytopathogenic fungi frequency was low in both crop cycles, its existence in this region is a potential risk, as these fungi affect the foliage, and, if the grain is contaminated, it can rot when stored, or cause the root and stem rot when the seed is used for planting.
Key words: Bipolaris sorokiniana; Helminthosporium sativum; Alternaria alternata; Septoria tritici; Fusarium proliferatum; Fusarium moniliforme
La producción de trigo (Triticum aestivum L.) bajo riego en la región de El Bajío, México, es afectada principalmente la roya de la hoja (Puccinia triticina Eriks.) y la roya lineal amarilla (Puccinia striiformis f. sp. tritici). En años recientes, en esta región se observaron otros síntomas como tizones y manchas foliares que también limitan la productividad de las variedades de trigo sembradas. Para identificar a los hongos asociados con esta sintomatología, en este estudio se recolectaron hojas con síntomas de tizones y manchas foliares en 12 municipios de los estados de Guanajuato, Michoacán y Jalisco, en los ciclos agrícolas otoño-invierno 2012-2013 y 2013-2014. La identificación de los hongos se realizó con la morfología, la amplificación y la secuenciación de las regiones espaciadoras transcritas internas (ITS). Los hongos fitopatógenos identificados fueron Bipolaris sorokiniana (Sacc.) Shoem. (Sinónimo: Helminthosporium sativum Pammel, C. M. King y Bakke), Alternaria alternata (Fr.:Fr), Septoria tritici, Fusarium proliferatum (Matsushima) Nirenberg, F. equiseti (Corda) Sacc., F. moniliforme (J. Sheld) (Sin. F. verticillioides (Sacc.) Nirenberg), Curvularia spp., Cladosporium spp., los saprófitos Nigrospora spp., Torula spp., Epicoccum spp., y el hongo antagonista Chaetomium globosum Kunze ex. Fr. Aunque la frecuencia de los hongos fitopatógenos fue baja en ambos ciclos agrícolas, su presencia en esta región es un riesgo potencial porque estos hongos afectan el follaje y al contaminar el grano del trigo pueden causar pudriciones cuando está en el almacén y pudriciones de la raíz y tallo cuando las semilla se usa para la siembra.
Palabras clave: Bipolaris sorokiniana; Helminthosporium sativum; Alternaria alternata; Septoria tritici; Fusarium proliferatum; Fusarium moniliforme
Introduction
In Mexico, wheat (Triticum aestivum L.) is sown on about 683 000 ha, producing 3.36 million t each year. The area sown with cereals ranks third after maize and sorghum. 3.17 million t are produced under irrigation, mainly in the States of Sonora, Chihuahua and Jalisco. The Bajio region, which includes the states of Guanajuato, Michoacan, Queretaro and Jalisco, is a geographic area the harvest 215 000 t under the same irrigation scheme (CANIMOLT, 2015; SIAP, 2015). Foliar diseases are considered the main limitation of crop yield. The leaf and stripe rust, caused by Puccinia triticina and P. striiformis f. sp. tritici, are the two major diseases, as well as the most studied, in irrigated wheat in the Bajio (Huerta et al., 2012; Solis et al., 2013; Mariscal, 2014). In this region, spots and leaf blights caused by unknown causative agents were observed, contributing to the reduced performance of the currently grown varieties. Zillinsky (1984) and Warham et al. (1999) indicated that Helminthosporium and Septoria genera cause these symptoms in wheat and are the most important. The species H. sativum, H. tritici-repentis, H. giganteum and H. spiciferum cause leaf blights in wheat worldwide. The fungus S. tritici is identified as the predominant species causing leaf spots. In Mexico, the presence of several species of Helminthosporium is documented (Osorio et al., 1998), as well as S. tritici on rainfed humid regions (Rodriguez et al., 2008). These fungi cause yield losses, between 4 % and 38 % (Acharya et al., 2011) and can be transmitted by the seed, causing stained grain, root rotting and seedling death (Zillinsky, 1984).
Our hypothesis was that the fungi that cause leaf spot and blight in wheat can be in any region where this cereal is sown and can cause significant yield losses. The aim was to identify the fungi associated with leaf spot and blight in the irrigated wheat area of the Bajio, in order to know its distribution and frequency in this area.
Materials and Methods
Collection of leaf tissue
The study included 10 wheat varieties sown in 16 locations in 11 municipalities in the States of Guanajuato, Jalisco and Michoacan, Mexico (Table 1). The varieties were sown in each locality during the Irrigation Wheat Program yield trials, from the INIFAP. The Cortazar S94 and Barcenas S2002 varieties are occupy the largest sown area at El Bajio (Solis et al., 2014). The samplings were made during the fall-winter agricultural cycles 2012-2013 and 2013-2014, at the milky-dough grain stage, close to maturity. The five of gold scheme was used, and on each point, a plot was selected, and of two furrows at random per plot, two plants with blights symptoms or leaf spots were selected. From them, a leaf with the symptoms was picked. This was carried out at each point (10 leaves total) in all sampled locations (160 leaves in total). The leaves were stored in glycine bags for their preservation, pressed and dried to prevent them from breaking (Zillinsky, 1984). The disease incidence was natural in all varieties and sampled locations, given the optimal temperature (25 °C) and relative humidity (80 %), along with the inoculum. That is because, all the evaluated sites are constantly sown with cereals, which is necessary for the development of fungi that infects wheat foliage (Narro, 2000; Marshall 2014).
Isolation and morphological identification of fungi
The samples were processed, and their morphological identification was performed at the Phytopathology Laboratory of INIFAP-CEBAJ; molecular techniques were performed at the Biotechnology Laboratory of the same institution. The collected leaves were processed following the Zillinsky protocol (1984). From each leaf, a central 8 cm length portion was cut and disinfected for 90 s with 5 % sodium hypochlorite. The portions were washed with sterile distilled water three times and allowed to dry on absorbent sterile paper. Two leaf pieces were placed in a wet chamber. These were Petri dishes (Ø=10 cm) containing sterile absorbent filter paper moistened with sterile distilled water. The chambers were then sealed with parafilm, labeled and incubated at 24 °C for 48 h before checking, afterward revisions were scheduled every 24 h depending on the fungi growth rate. The identification was made by the characteristics of the colonies, and direct observation of their conidia morphology in a stereoscopic microscope (Motic®, Model SMZ168TH, Motic, China). Also, temporary preparations with 10% Lactofenol were observed in a compound microscope (Motic®, Model BA410, Motic, China). The morphological identification was performed following the descriptors used by Leslie and Summerell (2006), Zillinsky (1984) and Warham et al. (1999). Portions of mycelium grown on the leaves were taken with a stereoscopic microscope and an entomological needle. For each growth, were cultured by duplicate four portions crosswise in Petri dishes (Ø=10 cm), in potato-dextrose agar culture medium (Bioxon®) and lactic acid (0.25 mL L-1)(PDA+LA). These were maintained at 24 °C. After 48 to 72 h, depending on the growth rate of each isolated mycelium, transfers were made by quadruplicate using the tip of hyphae technique into Petri dish with PDA+LA and maintained at 24 °C. After the fungus grew, a Petri dish was selected, a 5 mm2 portion of it was extracted and cultured by duplicate in Petri dishes (Ø=6 cm) with PDA + LA. From this dishes, pure cultures were obtained for molecular identification.
Molecular identification of fungi
Molecular identification of the pure cultures was performed by PCR amplification of the internal transcribed spacers (ITS) and sequencing and comparison with the GenBank database.
DNA extraction
The total DNA from each selected pure culture was extracted according to the protocol described by Lievens et al. (2003), with some modifications. Cultures of 5 to 10 d of age were used. The mycelium was obtained by scraping 2 cm2 sections from the margin of the colony and pulverized in a mortar with liquid nitrogen. The powdered mycelium was collected in 1.5 mL Eppendorf tubes, 300 µL of lysis buffer were added (2.5 M LiCl, 50 mM Tris, 62.5 mM EDTA and 4.0 % Triton X-100, pH 8.0) and an equal volume of phenol:chloroform:isoamyl alcohol mixture (25: 24: 1 v/v). Tubes were manually stirred for 30 s, centrifuged at 9300 g for 10 min (Sorvall microcentrifuge Legend ™ Micro 21R ™, Model LR58495, Thermo Fisher Scientific, Germany); 250 µL of the supernatant were then transferred to new 1.5 mL Eppendorf tubes, 15 µL of RNase were added (RNase A, Qiagen, Qiagen, 10 mg mL-1) and maintained at 37 °C for 30 min, to remove RNA. 300 µL of the phenol:chloroform:isoamyl alcohol mixture (25: 24: 1 v/v) were added to the tubes and manually stirred for 30 s, centrifuged at 9300 g for 10 min, and 200 µL of the supernatant were transferred to new 1.5 mL Eppendorf tubes. The total DNA was precipitated by adding two volumes of absolute ethanol and then incubating at -20 °C for 15 min. In order to compact the DNA, the tubes were centrifuged at 9300 g for 10 min, their supernatant was then decanted and the DNA pellet was washed with 200 µL of 70 % ethanol and centrifuged at 11,200 g for 5 min. The supernatant was decanted and the DNA pellet was allowed to dry for 3 h. The pellet was resuspended in 30 µL of sterile deionized water. Genomic DNA was quantified in a spectrophotometer (Nanodrop 8000, Thermo Scientific®, Model ND-8000, Thermo Fisher Scientific, Germany) at 260 and 280 nm, and its integrity was checked by electrophoresis in 1 % agarose gels. Then, all DNA samples were diluted to 10 ng µL-1 for PCR amplification of the ITS.
PCR Amplification of ITS
The forward primer ITS1-F: 5’-CTTGGTCATTTAGAGG AAGTAA-3 ‘(Gardes and Bruns, 1993) and reverse ITS4: 5’-TCCTCCGCTTATTGATATGC-3’ (White et al., 1990) were used to amplify the ITS. ITS1 Primers delimit the region 5.8 S and ITS2 from fungi. The polymerase chain reaction consisted of 25 ng of template DNA, 1x PCR buffer (SENNA®), 0.2 mM of each dNTP, 0.5 µM of forward primer, 1.0 µM of the reverse primer and 1 U Taq High Fidelity DNA polymerase (SENNA®) in 25 µL of total volume. Amplifications were carried out in a thermocycler (T100™ Thermal Cycler, Bio-Rad, Model T100, USA) and initial denaturalization of 94 °C for 2 min, followed by 35 cycles at 94 °C for 45 s, 59 °C for 45 s 72 °C for 90 s, and a final extension of 72 °C for 10 min. The amplicons were analyzed by electrophoresis in 1 % agarose gels and photo-documented with Gel Logic 212 Pro (Carestream®, Model GL 212 Pro Imaging System, USA).
Purification and sequencing
The amplified fragments were cut from the Agarose gel with a scalpel, and purified with a Freeze’N Squeeze DNA Gel Extraction Spin ™ Columns (Bio Rad®-) kit following manufacturer instructions. The amplicons were sequenced in the Department of Genomic Services LANGEBIO, CINVESTAV-Campus Guanajuato. The sequences were compared using the GenBank database and the Nucleotide BLAST tool (NCBI, 2015).
Fungi frequency calculation
The frequency of fungi per municipality (FM) was calculated by grouping the two evaluated agricultural cycles; the overall frequency (OF) of each agricultural cycle was adjusted using the formulas proposed by Iftikhar et al. (2006). For FM, we used the formula FM (%)=(Num. Times the fungus was identified in the total leaves collected per municipality in both cycles / total mycelial growth examined in all leaves) (100). For FG, the formula IG (%)=(Num. Times the fungus was identified on the collected leaves by agricultural cycle / total mycelial growths analyzed in leaves) (100).
Results and Discussion
Morphological identification
The identified fungi belonged to the Alternaria, Cladosporium, Chaetomium, Epicoccum, Torula, Curvularia, Nigrospora, Fusarium, Helminthosporium and Septoria genera. In the Alternaria genus, short four to seven conidia chains were observed, with light brown coloration, ovoid and smooth-walled, with several transverse septa (Figure 1A) and 18 to 36 µm x 10 to 15 µm in size. These characteristics are consistent with those described by Zillinsky (1984) and Warham et al. (1999) for species of this genus. Cladosporium conidia developed in bush-like groups of gray color (Figure 1B), with branched conidiophores and ovoid conidia, lightly brown and mostly with one or two septa, 8 to 20 µm x 3 to 11 µm (Figure 1C) (Zillinsky, 1984; Warham et al. 1999). Epicoccum presented as masses of conidia with dark brown to black coloring (Figure 1D), the majority it spherical without visible septa, rough wall and diameter of 13 to 20 µm; these characteristics coincided with those described by Warham et al. (1999). On the lesions, spherical and elongated perithecia of Chaetomium were observed (Figure 1E). At the perithecis, unicellular oval ascospores were observed (Figure 1F). In Torula conidial chains with spherical unicellular conidia were observed with light brown color, approximate 5.5 to 6 µm in diameter (Zillinsky, 1984; Warham et al. 1999). The fungus Nigrospora presented short hyaline conidiophores with light brown coloration, in right angle, conidia were dark brown to black, the majority spherical (Figure 1G) with a diameter of 13 to 15 µm (Warham et al. 1999).
![](/img/revistas/agro/v51n2//1405-3195-agro-51-02-00189-gf3.jpg)
Figure 1 Identified fungi from blights and leaf spots in irrigation wheat. Conidia of Alternaria spp., (A).; conidia cluster (B) and individual (C) of Cladosporium spp.; Epicoccum spp., conidia (D).; perithecium (E) and ascospores (F) of Chaetomium spp.; conidia of Nigrospora spp., (G).; conidia of Curvularia spp., (H).; micro (I) (Mi) and macroconidia (I) (Ma) of Fusarium spp.;conidia of Helminthosporium spp., (J).; symptoms and pycnidia (Pi) of Septoria tritici grown on leaves (K).
Individual conidiophores were observed in Curvularia of light brown coloration with several septa, conidiophores were straight with no ramification (Figure 1H); the conidia were curved with four visible septa, light brown coloration when young and darker brown as they aged. The central cell of the conidia was larger and darker than the others (Figure 1H) (Zillinsky, 1984; Warham et al., 1999) in Fusarium, hyaline microconidia were observed, the most straight, with 1 to 2 septa of 9 to 22 µm x 3 to 6 µm (Figure 1I). The macroconidia were slightly curved with 3 to 4 septa and with a curved apical cell of 25 to 42 µm x 3 to 5 µm (Figure 1I) (Leslie and Summerell 2006). In Helminthosporium individual conidia with conidiophores in clusters and dark brown to black coloring were observed. There were two types of conidia (Figure 1J), a straight, single, slightly wider in the middle, with brown-olive colour to light brown, 6 to 10 septa with rounded tips and 60 to 78 µm x 15 to 20 µm. The other conidia shorter, oval, with more rounded ends and wider in the middle, with 6 to 8 septa from 55 to 73 µm x 12 to 17 µm (Zillinsky, 1984; Warham et al. 1999). Septoria tritici was identified based on the signs and symptoms only of the leaf injuries; these were irregular patches of dark brown coloration with typical pycnidia development (Figure 1K) (Zillinsky, 1984).
Molecular identification
Molecular identification was made in Curvularia spp., Fusarium spp., Chaetomium spp., Helminthosporium spp., and Alternaria spp., given that they presented morphological differences in the pure cultures, as in their mycelium color, type of hyphal growth, pigmentation of the culture medium and conidia morphology. These could be due to the presence of more species. The six obtained sequences were stored in the GenBank database (NCBI, 2015) with access numbers KR819408, KR819405, KR819409, KR819406, KR819404 and KR819407. BLAST comparison at the KR819408 (383 bp) sequence had a 97 % similarity with F. proliferatum (GenBank access No. KJ439117). KR819405 (763 bp) had a 100 % similarity with F. equiseti (GenBank access No. HQ718414). KR819409 (262 bp) was a 100 % similar to JF499675 Gibberella moniliformis sequence (Anamorph: F. moniliforme, synonym F. verticillioides). KR819406 (877 bp) was 97 % similar to Chaetomium globosum (GenBank access No. KP067226). KR819404 (970 bp) was 99 % similar to the sequence KP174682 of B. sorokiniana (synonym: H. sativum) and KR819407 (973 bp) showed 99 % similarity to Alternaria alternata (GenBank access No. KJ867625). A sequence of Curvularia spp., of 148 bp, was 100 % similar to sequences of Cochliobolus lunatus and C. geniculatus (GenBank access No. JX868663 and KF946043), teleomorphs of C. lunata and C. geniculata; but no access number was assigned given that their sequence was less than the 200 bp indicated by GenBank. The molecular characterization confirmed the morphology identification and allowed the identification of the species by taxonomic keys.
Fungi frequency
Chaetomium spp., was the most frequently isolated in the two crop cycles and in the majority of the assessed municipalities (Table 2) (Figure 2); it is considered a saprophyte (Warham et al., 1999) and the cause of wheat grain discoloration (APS, 1987). Chaetomim globosum is not reported to cause leaf spots or blights; but it has shown antagonistic activity against B. sorokiniana, S. tritici and A. triticimacularis (Perello et al., 2002; Aggarwal et al., 2004). Therefore, its high frequency in the affected foliage in our study could be due to the fact of its activity as an antagonistic microorganism.
Table 2 Fungi identified in wheat varieties with symptoms of leaf spots and blights; frequency and percentage by municipality. Fall-winter 2012-2013 and 2013-2014 agricultural cycles
![](/img/revistas/agro/v51n2//1405-3195-agro-51-02-00189-gt4.jpg)
NHR/NCME: No. of collected leaves/No. examined mycelial growth
![](/img/revistas/agro/v51n2//1405-3195-agro-51-02-00189-gf4.jpg)
Figure 2 General frequency of fungi identified in wheat leaves with leaf spots and blights symptoms collected at the Bajio, during fall-winter 2012-2013 and 2013-2014 agricultural cycles.
The genus Alternaria was the second most frequent and it appeared in more than 30% of the grown mycelia, in both of the agricultural cycles analyzed (Figure 2). The species A. alternata, identified in this study, has caused leaf blights that have generated considerable losses in wheat and rice elsewhere (Iram and Ahmad, 2005; Iftikhar et al., 2006). This fungus can be dangerous for oat crops in the Central Mexican Plateau (Garcia et al., 2013). Bipolanis sorokiniana (syn. H. sativum) is causing leaf blight in wheat and is considered one of the most destructive fungi for this cereal in some areas of the world (Acharya et al., 2011), which has cost 20 to 60 % of yield losses (Duveiller et al., 2007; Rattu et al., 2011). In five municipalities in Guanajuato (Table 2) B. sorokiniana (syn. H. sativum) was identified, but with a low frequency (Figure 2), probably because the conditions were not optimal along with the antagonistic effect of C. globosum. Septoria spp., was identified as Septoria tritici because the collected plants showed symptoms only in the leaves, in contrast to S. nodorum that also it infects the glumes (Eyal et al., 1987). Septoria tritici is the species responsible of the leaf spot and it causes 44 % yield losses in Mexico (Rodriguez et al., 2008) and 12 % in irrigated wheat in other countries (Rezgui et al., 2008). This fungus was identified only in the Michoacan and Jalisco municipalities. It had low frequency, similar to that of B. sorokiniana. This is the first report of these three fungi on irrigated wheat at the Bajio. Several species of Fusarium that cause lesions on leaves in humid environments are also documented (APS, 1987); F. Equiseti, F .proliferatum, and F. moniliforme were identified in infected wheat leaves in the United States (Ali and Franci, 2001), and F. equiseti in young leaves of cereals in the Mexican high valleys (Zillinsky, 1984); these are not considered an important cause of blights. But the three species are pathogenic, cause wheat scab (Chehri et al., 2011) and can synthesize mycotoxins and cause root rots (Goswami et al., 2008); because of this, the pathogen in optimal conditions could be a danger to the crops.
The fungus Curvularia spp. was identified in two municipalities (Table 2) with low frequency (Figure 2). This was identified as the cause of leaf spots in wheat (Warham et al., 1999), sorghum and maize (C. lunata); and in maize it caused up to 60% of losses (Akinbode, 2010). Although it was observed that Cladosporium spp. is more frequently associated with black mold or soot of wheat ear (Warham et al. 1999; APS, 1987), Smiley et al. (1993) isolated with a high frequency three species of this genus from foliar injuries to assess a disease called “physiological leaf spot” in this cereal. The frequency of the Torula spp., Epicoccum spp., and Nigrospora spp., fungi which are considered saprophytes (Warham et al. 1999), was low. Although Nigrospora spp., is one of the causes for the black spot in wheat grain (APS, 1987), in rice it was isolated from leaf lesions (Hajano et al., 2011). Torula spp., and Epicoccum spp., also cause black mold and spots in wheat (Warham et al. 1999; APS, 1987), but in our study it seems that they were were as secondary invaders in leaf lesions caused by other fungi (Zillinsky, 1984).
Conclusions
In the Bajio, Mexico, the B. sorokiniana, A. alternata and Septoria tritici fungi were identified in spots and leaf blights of wheat. F. proliferatum, F. verticillioides, F. equiseti, Curvularia spp., Cladosporium spp., and Nigrospora spp., were also identified. Although the frequency of most phytopathogenic fungi was low, its impact on all sown varieties and the movement between regions of contaminated seeds, could affect any production area of this cereal.
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Received: October 2015; Accepted: November 2016