Introduction

In Mexico, 9966 ha were planted with strawberry (Fragaria×ananassa Duch.) during 2014. Production from the states of Michoacan (259 000 Mg), Baja California (146 000 Mg) and Guanajuato (29 000 Mg) was 94 % of the Mexican production. In that year, the value of the fruit production in the state of Guanajuato was 124 million pesos (9.3 million US dollars) (^{Servicio de Información Agroalimentaria y Pesquera, 2015}). In this state, the crop is susceptible to fungal diseases in root and crown, so plantings area decreases each year. Strawberry dry wilt is the most common disease and generates the gradual death of the plant (^{Castro and Dávalos, 1990}).

In the central Mexico area, Fusarium oxysporum causes the disease, is the most common fungus because it occurs from early stages, and cause losses greather than 50 %, equivalent to 7.5 Mg ha^-1 (^{Dávalos et al., 1992}). Along with this species, F. solani, F. moniliforme (sin F. verticillioides), Phytophthora fragariae, Rhizoctonia solani, R. fragariae, Alternaria spp., Pythium aphanidermatum, Verticillium alboatrum, Colletotrichum spp., Cylindrocladium spp., and Ramularia spp., have also been identified as part of the problem and cause significant losses depending on the environmental conditions, crop management and soil texture (^{Castro and Dávalos, 1990}; ^{Ceja et al., 2008}).

Analyzes of plants with typical symptoms of strawberry dry wilt at the Phytopathology Laboratory of the INIFAP-CEBAJ, culture and morphological differences in Fusarium isolates suggested that more than one species or different species to those reported, were present and caused the observed disease. The fungus Alternaria spp. was also isolated more frequently than other more important fungi, such as Verticillium spp., and Rhizoctonia spp., but it is not yet identified to a species level (Mariscal, 2015^[3]). Therefore, the objective of this study, was to assess the current presence of the fungi associated with the disease, to estimate their frequency and identify it through cultural and morphological characteristics, and sequencing of ITS regions, but also identify other species associated with this phytosanitary problem in Guanajuato. Our hypothesis was that there are other species of fungi, different to those reported so far, that cause the strawberry dry wilt.

Materials and Methods

During the spring-summer 2014 (PV/2014) and 2015 (PV/2015) growing season, plants of the ‘Camino Real’, ‘Festival’, ‘Albión’, ‘Portales’, ‘Camarosa’ and ‘San Andrés’ cultivars were collected. These had characteristic strawberry dry wilt symptoms (wilting, dwarfing and chlorosis). They were collected in 34 commercial plots located in three municipalities in the state of Guanajuato. From the 170 samples, 160 were from Irapuato, five from Abasolo and of five from Salamanca. Fungal isolates were purified using the monospore cultures and hyphal tip technique in Potato-Dextrose-Agar media (PDA, BD Bioxon®), 39 g L^-1 distilled water with a 0.25 % lactic acid addition. From each plant, eight pure isolates were obtained, four from the root and four from the crown tissue.

The first identification was made from 511 pure isolates obtained from the PV/2014 and 833 from the PV/2015 cycle. For the Fusarium genus classification, the mycelial growth and culture media pigmentation was observed; microconidia, macroconidia and chlamydospores characterization was performed by temporary preparations. Culture and morphological characteristics were compared with ^{Nelson et al. (1983)} and ^{Leslie and Summerell (2006)} descriptions. For the other fungi genera, the growth type, mycelium color and the shape and size of their conidia were compared with the descriptions of the ^{University of California (1994)} and ^{Anaya and Romero (2011)}. Due to the high number of isolates classified in the Fusarium genus, one isolate from each location was selected and cultured in Carnation Leaf Agar (CLA) (HYCEL®), 20 g L^-1 distilled water. For better comparison, the certified stain of F. oxysporum f. sp. fragariae [IFO 31180 (8)] (ATCC®) was cultured in PDA and CLA.

The selected isolates were used for molecular identification by amplifying and sequencing the internal transcribed spacers (ITS). DNA extraction of each isolate was performed adapting the ^{Lievens et al. (2003)} protocol. For the PCR the ITS1-F oligonucleotide (direction 5’-CTTGGTCATTTAGAGGAAGTAA-3’) (^{Gardes and Bruns, 1993}) and the antisense ITS4 oligonucleotide (5’-TCCTCCGCTTATTGATATGC-3’) (^{White et al., 1990}) which delimit the ITS1, the 5.8 S and the ITS2 region of the fungus were used. The reaction mixture was 25 ng DNA, 1x PCR regulator (SENNA®), 0.2 mM of each dNTP, 0.5 µM of the sense oligonucleotide, 1.0 µM of the antisense oligonucleotide and 1 U Taq High Fidelity DNA Polymerase (SENNA®) in a 25 µL total volume. Amplification was done in a T100 thermocycler (Bio-Rad®) with the program: 1 cycle at 94 °C, for 2 min; 35 cycles at 94 °C, 45 s; 59 °C, 45 s; 72 °C, 90 s; 1 cycle at 72 °C, for 10 min. The products were purified with a Freeze ‘N Squeeze™ kit DNA Gel Extraction Spin Columns (Bio-Rad®) following the manufacturer’s protocol. The purified products were sequenced at the Genomic Services Laboratory LANGEBIO, CINVESTAV-Guanajuato Campus, using the Sanger method. The obtained sequences were compared and aligned with the NCBI, USA, GenBank database, with the Nucleotide BLAST (^{National Center for Biotechnology Information, 2015}).

The isolation frequency of the fungi genera by agricultural cycle was calculated using the equation: Frequency (%)=(Number of isolates from the identified genus / total isolates analyzed per cycle)*100 (^{Juber et al., 2014}).

Results and Discussion

The results of the fungi identification and their isolation from root and crown infected tissue from strawberry plants revealed the presence of three major genera already reported by ^{Castro and Dávalos (1990)} and ^{Ceja et al. (2008)}. The most frequent genus was Fusarium isolated from all plants from all locations in both evaluated agricultural cycles. The frequency of this genus was 54 % in the PV/2014 and of 60 % in the PV/2015 (Figure 1). These results match the high frequencies reported by ^{Castro and Dávalos (1990)} and Brow et al. (2008), who isolated Fusarium spp., in 47 % and 83 %, repectively. In PDA, this microorganism showed different culture characteristics such as white, pink, salmon, red, magenta and violet pigmentations, which became darker with age, and in some cases, abundant cottony mycelium scattered along the Petri dish. Pigmentations in CLA did not occur in any isolates but, yellow, creamy yellow and pale orange sporodochia appeared over the carnation leaves. By microscopic analysis abundant hyaline and ovoid to ellipsoidal microconidia were detected, with 0-1 septa (Figure 2J) of 1.8-3.8×2.06-12.0 µm. At least four types of macroconidia were observed of 1.68-4.0×12.4-41.5 µm with 3-4 septa (Figure 2B-I). Most macroconidia were hyaline, curved with a curve apical cell and a foot-shaped basal cell (Figure 2E), some with similar macroconidia characteristics of F. oxysporum f. sp. fragariae [IFO 31180 (8)] (ATCC®) measuring 1.68-2.60×17.70-30.60 µm (Figure 2A). Some isolates produced abundant terminal chlamydospores or interleaved in the mycelium, solitary or in 3-7 cell chains with roughened warty cell walls (Figure 2K, L). These culture and morphological characteristics coincided with those described by ^{Nelson et al. (1983)} and ^{Leslie and Summerell (2006)} for species of this genus and in some isolated for F. oxysporum.

Figure 1 Frequency in the percentage of Fusarium spp., Alternaria spp., and Rhizoctonia spp., in 511 isolates from root and crown infected tissue of strawberries from the 2014 spring-summer growing season and 833 isolates from the 2014 spring-summer season. 

The genus Alternaria was the second most common in both cycles: 36 % in the PV/2014 and 29 % in the PV/2015 (Figure 1). These frequencies partially coincided with those reported by ^{Castro and Dávalos (1990)} where Alternaria spp. was isolated in 5 % to 12 % and the third most common fungus after Fusarium spp., and Verticillium spp. In PDA, the isolates showed a white color cottony mycelial growth which turned gray and dark brown with age. Two types of conidia were observed, the first were short, in 3-5 cell chains, often with three septa, ovoid, light brown, with smooth walls and a short conical apical cell (Figure 2N). The latter, were longer conidia in 6-8 cell chains, often with six septate, brown clear, smooth walls and a conical apical cell (Figure 2O), of 4.0-8.8×9.6-5.22 µm. The characteristics of the first conidia (Figure 2N) were similar to those described by ^{Simmons (2007)} for Alternata. In other studies, Alternaria spp. was isolated from infected roots and crowns, but this species did not cause infection in these parts of the plant.

Rhizoctonia was the less frequent genus with 11 % in both agricultural cycles (Figure 1); ^{Castro and Dávalos (1990)} and ^{Ceja et al. (2008)} also reported this genre as less frequent with 2-15 % and 2-10 % each, respectively, which coincides with the low frequencies calculated in our study. Isolates in the PDA showed a slightly ringed mycelial growth with white to light brown mycelium. The fungus produced typical hyphae with branches in right angles (90 °C), with a slight constriction of the hyphae at the beginning of the branch and the two distinct septa, one in the main hyphae and the other in the branch (Figure 2P). Hyphae staining with safranin indicate the presence of multinucleated hyphae, which correspond to the R. solani species (Figure 2Q) (^{UC, 1994}; ^{Anaya and Romero, 2011}).

Amplicons obtained with the sense oligonucleotide ITS1-F and the antisense ITS4 oligonucleotide, from DNA of the selected Fusarium isolates were approximately 600 to 850 bp (Figure 3). The 34 ITS sequence from the selected isolates was obtained. Due to its size (>200 bp), only 15 sequences were stored in the GenBank; nine of them (accession numbers: KT183399, KT183401, KT183402, KT183404, KT183405, KT183406, KT183408, KT183409 and KT1834-10) had a 99-100 % homology with most sequences of F. oxysporum. The sequence KT183403 had a 100 % homology with the F. oxysporum and F. oxysporum f. sp. ciceris species. The last five sequences (KT183400, KT183407, KT183411, KT183412 and KT183413) had homology percentages with F. verticillioides (100 %), F. pseudonygamai (100 %), F. fujikuroi (100 %), F. proliferatum (100 %) and F. brachygibbosum (99 %). By association between the two identification techniques and characteristics of the macroconidia of F. oxysporum f. sp. fragariae (Figure 2A), macroconidia in Figure 2B, C, D and E, corresponded to F. oxysporum. Macroconidia (Figure 2G) and the long chains of microconidia (Figure 2M) correspond with the characteristics from F. verticillioides; the macroconidia (Figure 2H) and the culture characteristics of the isolates from which they were extracted, matched those of F. proliferatum (^{Nelson et al., 1983}; ^{Leslie and Summerell, 2006}; ^{Pitt and Hocking, 2009}).

Figure 2 Micrographs of Fusarium spp.; different types of macroconidia (A-I); microconidia (J); chlamydospores (K-L); microconidia in long chains (M); Alternaria spp., with two types of conidia (N-O), and Rhizoctonia spp., hyphae with 90° (P) and multiple cores (arrows) (Q). 

Figure 3 Amplification of ITS isolates from Fusarium spp., with the ITS1-F direction oligonucleotide and ITS4 antisense oligonucleotide; M=marker of 1 kb size Plus DNA Ladder (Thermo Fisher Scientific®); lane 1-13, Fusarium spp., samples; the amplified fragments are shown with an arrow. 

Different species of Fusarium identified in our study indicate that strawberry dry wilt is caused by a species complex; these were identified and isolated from infected root and crown tissue and include: F. solani (^{Ceja et al., 2008}), F. verticillioides (^{Téliz et al., 1986}) and F. oxysporum (most common) (^{Castro and Dávalos, 1990}). However, it is necessary to tests the pathogenicity with other species of this genus molecularly identified: F. pseudonygamai, F. fujikuroi, F. proliferatum, and F. brachygibbosum, which have not been reported to cause disease in the crop, unlike A. alternata. The species of F. solani, Cylindrocarpon spp., Pythium aphanidermatum, Phytophthora sp., Verticillium spp., Phoma spp., and Colletotrichum spp., reported by ^{Castro and Dávalos (1990)} and ^{Ceja et al. (2008)} in Guanajuato, were not identified in our study.

Conclusions

The most abundant genera are Fusarium, Rhizoctonia and Alternaria of which Fusarium oxysporum from the Fusarium species is the most common. The second and third most common genre were Alternaria, with the species A. alternata and Rhizoctonia, isolated and identified by their morphology. The various culture and morphological characteristics along the comparison and identification by ITS of seven Fusarium species suggest that strawberry dry wilt is caused by a complex of different species and other fungi not reported