Strawberries (Fragaria spp.) are one of the most widely consumed fruits in the world, due to their high contents of flavonoids, anthocyanins, phenolic compounds and vitamins A and C (^{Cao et al., 2011}; ^{Cano, 2013}). For the Mexican economy, cultivating strawberries is an important source of income; Mexico is the country with the third highest export, with 102,631 t a year, only after Spain and the United States (^{FAOSTAT, 2016}). In Mexico, in the state of Guanajuato beginning in 1950, the crop became more important due to the increasing demand from the United States, which led it to expand to other states, such as Michoacán and Baja California, where they went from covering the needs of the local market to being large producers of strawberries in Mexico (^{Ávila and González, 2012}). At a national level, strawberry production is exponential; last year, the fruit was planted in 13,564 ha, with a production of 611,780 t, with the most important states being Michoacán with 9,837 t, Baja California with 1, 920 t and Guanajuato with 1,197 t (^{SIAP, 2018}). The state of Morelos is beginning to plant more than 8 ha of the crop, although one of its main problems in the management of the diseases that affect it, which are mostly fungal, followed by some bacterial problems, nematodes and others caused by viruses (^{Martínez et al., 2010}).

Strawberry dry wilt is considered the most destructive disease for this crop, due to its incidence, distribution and difficult control, since it leads to the gradual death of the plant (^{Ceja et al., 2008}). The disease has been related with several species of Fusarium, Verticillium dahliae, Rhizoctonia solani and Phytophthora spp. (^{Castro and Dávalos, 1990}; ^{Quintero et al., 1998}). In the central area of Mexico, Fusarium oxysporum causes the disease, and it is the most frequent fungus, because it attacks in early stages of the crop and it causes losses of above 50% (^{Dávalos et al., 1992}). However, in the state of Guanajuato, 14 species of fungi have been reported, and F. oxysporum is the most frequent. Despite having studies on the disease in central Mexico, in the state of Morelos there are problems with strawberry dry wilt, but no reports of the causal agent, hence the importance of this investigation, with the aims of determining the pathogen using morphological identification, as well as carrying out pathogenicity tests and comparing with a reported species, evaluating inoculation methods and determining the damage caused on the root.

In September 2018, using a random selection, 50 samples of stems and roots with symptoms of dry wilt were collected in a commercial plantation of the Alexandria variety, the most predominant, in different stages of the crop, located between coordinates 18° 52′ 59″ Norte, 99° 4′ 1″ West in the plot of Chapultepec, municipal area of Yautepec, Morelos. The samples were taken in an ice chest to the Phytopathology laboratory of the Escuela de Estudios Superiores de Xalostoc (EESuX). In order to isolate the causal agent, 50 fractions of affected tissue were taken: 0.5 cm² of roots and stems. They were disinfested with sodium hypochlorite at 1% for 3 minutes, followed by three washes with sterile distilled water and they were left to dry on sterile paper towels. They were planted in a Potato Dextrose Agar (PDA) medium and incubated at a temperature of 25 °C for 7 days. After sporulating, monosporic cultures were carried out, and using the hypha tip technique they were moved to Petri dishes with PDA culture medium. The pathogen was identified by coloring the colony, type, size and shape of the spores, and we used the features described by ^{Nelson et al. (1983}) and ^{Leslie and Summerell (2006}) for species of this genus and it was compared with isolations of the strain (IFO 31180 (8)) (ATCCÒ) of F. oxysporum obtained from the INIFAP, originating from the municipality of Celaya, Guanajuato, since it presented the same symptoms and structures of the pathogen described by ^{Mariscal et al. (2017}).

In order to corroborate that the isolation, present in 95% of the samples, is the cause of the strawberry dry wilt, and to evaluate its severity, pathogenicity tests were carried out. For this, the isolation of the fungus was increased in eight flasks with 200 g of oat, previously saturated in water for 24 h and sterilized. In four flasks with oats, we placed PDA discs with mycelial growth of the pathogen and in the four remaining flasks, we planted the comparative strain of F. oxysporum (IFO 31180 (8)) (ATCCÒ). The eight flasks were left to incubate at a temperature of 25°C for 15 days until the pathogen invaded the oat. For the inoculation of the pathogen, we took 20 plants from stolons from a greenhouse, which were grafted in a sterilized substrate, and after one month, they were inoculated using different methods, making up an experimental design totally at random with five treatments and four repetitions: T1= Inoculation of the root with infected oat (100 g plant^-1) by the Morelos strain at the moment of grafting, T2= Inoculation of the root with infected oat (100 g plant^-1) by the Celaya strain at the moment of grafting, T3=Immersion of the root a suspension of 1 x 10^-6 conidia mL^-1 of the Morelos strain before grafting, T4= Immersion of the root in a suspension of 1 x 10^-6 conidia mL^-1 of the Celaya strain before grafting and T5=Control (inoculation of the root with oat in the absence of the fungus). All plants were grafted into 10-inch plastic pots; they were added the substrate of peat moss + worm-compost in a ratio of 2:1 to give the plant the optimal conditions and they were then placed in a greenhouse. Thirty days after inoculation, an evaluation was carried out on the degree of severity of the disease in the entirety of the plants, and for this, we used an arbitrary scale of five types, designed by the authors of the present study (Table 1). Afterwards, root samples were taken to re-isolate the pathogen of each inoculated treatment.

Variation was found in the symptoms displayed by the plants in the aerial section, and we deduced that something similar was taking place in the root. Due to this, the roots of each plant were analyzed after washing to remove the soil from them, and next, the fresh weight of the plants was established using an analytical scale of the brand Scout-Pro, Mexico. The same plants were placed in paper bags and moved into a drying furnace, where they were kept for 24 hours at a temperature of 80 °C to establish their dry weight. The data for fresh and dry weights of the plants underwent an analysis of variance and Tukey’s test (α=0.05) using the statistical analysis package SAS^® version 9 for Windows.

The Morelos isolation displayed chlamidospores of three cells that were observed in chain, as well as the measurement of 50 oval-shaped microconidia with an average size of 9.44 μm in length and 3.46 μm wide. The macroconidia presented a foot-shaped basal cell and an average size of 24.20 μm in length and 4.19 μm wide (Figure 1A and 1B). These structures coincided with the comparative strain of Fusarium oxysporum (IFO 31180 (8)) (ATCCÒ) and both coincided with the characteristics described by ^{Nelson et al. (1983}) and ^{Leslie and Summerell (2006}) for the genus Fusarium. Regarding the coloring of the colony, both developed initially colored white and a cottonlike appearance, and as they expanded, they turned lilac, similar to the characteristics described by ^{Garcés et al. (2001}), which obtained strains of Fusarium with abundant aerial mycelia, cottonlike, with a variable color, from white to peachy-pink, but usually with a more intense purple or violet color on the surface of the agar (Figure 1 C and D).

Figure 1 Morphological characteristics of Fusarium oxysporum. A) microconidia and macroconidia of the Morelos strain, B) Microconidia of the Celaya strain, C) colony of the Morelos strain D) colony of the Celaya strain. 

The two Fusarium isolations used to inoculate the strawberry plans, both by applying infected oat directly on the root and by its immersion in the suspension of conidia, were 100% pathogenic 30 days after inoculation, where all the infected plants displayed evidence of damage located in levels two and three in the scale of severity of the disease. The strawberry plants inoculated with both isolations of Fusarium displayed typical symptoms of vascular wilting, yellowing and drying of the foliage. Reduction and necrosis of roots was observed, in comparison with the control, which presented none of the typical symptoms of the disease. These results may suggest the moment of the greatest infection of dry wilt within the phenology of the crop, and may therefore be the starting point for the control program of the disease. Results similar to those of the present study were obtained by ^{Ceja et al. (2008}), when 100% of the strawberry plants from the Camarosa cultivar began showing symptoms after 23 days, and wilted 30 days after inoculation with F. oxysporum. The fungi that caused the disease and that were re-isolated and compared morphologically displayed coincidences in their characteristics, fulfilling Koch’s postulates.

The values for fresh weight and dry weight of the plant displayed the negative effect of strawberry dry wilt on the growth of the crop. Both for fresh and dry biomass, there were significant differences between T5 (control) and the treatments under inoculation with the pathogen. The fresh weight of the plants in T5 reached 168.8 g, whereas the weights of the plants in the inoculated treatments (T1, T2, T3 and T4) went no higher than 81.23 g and the root appeared completely necrotized. A similar result was obtained for dry biomass; T5 displayed the greatest dry weight with 65.88 g and the remaining treatments only reached 26.73 g. The isolation of the Morelos F. oxysporum strain showed the same effects than the Celaya F. oxysporum strain for fresh and dry weight of the plant. There were no significant differences in these parameters with the use of one or other method of inoculation, either, which confirmed the effectiveness of both methods (Figure 2). These results confirm that the plants infected by Fusarium display a slow growth and the production of biomass is reduced significantly; F. oxysporum causes chlorosis, epinasty, defoliation and wilting due to damage in the vascular system of the host. Wilting takes place as a consequence of the obstruction of the plant’s vascular system due to the production of spores; the destruction of the vascular system due to the action of enzymes; the production of toxins, phenols and polysaccharides by the fungus, and the effect of the formation of tyloses (^{Agrios, 1997}). In cases of severe infections, plants die three and four weeks after infection (^{Lira and Mayek, 2006}). However, in the present study, symptoms were not very severe in some plants, since the plant grew roots, although the infection was severe.

Figure 2 Effect of the inoculation of the strain of F. oxysporum on the fresh weight and the dry weight of the plant. IAv: Inoculation of oat. IISC: Inoculation by immersion in a suspension of conidia. Different letters for a same variable differ by Tukey (p≤0.05). 

We conclude that the morphological characteristics of the isolated Morelos strain displays F. oxysporum as the causal agent of dry wilt in strawberry. Pathogenicity tests pointed at this fungus as the cause of the symptoms of vascular wilting, yellowing and root necrosis, as well as the reduction in fresh and dry biomass in strawberry plants. These results set the standards to guiding a control and for future investigations aimed at the search for molecular identification and the genetic and pathogenic relations of their populations.