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).

Table 1 Municipalities, number of locations and sampled wheat varieties in wheat fields in the Bajio during he agricultural cycles fall-winter 2012-2013 and 2013-2014 

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.

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}).

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 

NHR/NCME: No. of collected leaves/No. examined mycelial growth

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}).