Introduction

The tomatillo (Physalis ixocarpa Brot. ex Horm.), also called husk tomato or green tomato, is one of the most widely cultivated horticultural crops in Mexico, and more than 60% of the production is concentrated in the Mexican states of Sinaloa, Zacatecas, Jalisco, Puebla, Michoacán, and Sonora (SIAP 2018). In 2017, Sinaloa was the first largest producer of tomatillo in Mexico with a production of 150,697 t (SIAP 2018) with the main production area in this state is located in the northern region, including the municipalities of Guasave, Ahome, and El Fuerte.

In Mexico, the tomatillo plants are commonly affected by several plant pathogens including fungi (Apodaca-Sánchez et al. 2008), viruses (Méndez-Lozano et al. 2001; De La Torre-Almaráz et al. 2003; Gámez-Jiménez et al. 2009; González-Pacheco and Silva-Rosales 2013), and phytoplasmas (Santos-Cervantes et al. 2006; Mauricio-Castillo et al. 2018). The most important fungal diseases found in tomatillo fields in Mexico are white smut (Entyloma australe), white mold (Sclerotinia sclerotiorum) (Apodaca-Sánchez et al. 2008), leaf spot (Cercospora physalidis) (Félix-Gastélum et al. 2007; Apodaca-Sánchez et al. 2008), powdery mildew (Podosphaera xanthii) (Félix-Gastélum et al. 2007), leaf blight (Alternaria sp.) (Soto et al. 1998), and wilting (complex of soilborne fungi) (Apodaca-Sánchez et al. 2008).

Tomatillo wilt is a serious disease that commonly occurs wherever the crop is cultivated in Mexico and has been associated with a complex of soilborne fungi, such as Fusarium oxysporum, Neocosmospora solani (syn. Fusarium solani), Rhizoctonia solani, Pythium sp., Macrophomina phaseolina and Sclerotium rolfsii. The first visible symptom of the disease is yellowing of the foliage that is followed by poor growth, wilting, and death of plants. On roots, a light to dark brown rot is observed, which sometimes extends towards the neck and base of the stem. In diseased plants, the number and size of fruits are smaller, and they fall easily. The disease can occur at any stage of crop development, with economic losses that can exceed 50%. This disease is very important because tomatillo cultivars are highly sensitive to this fungal complex and the disease management is difficult and expensive using cultural and chemical methods (Apodaca-Sánchez et al. 2008).

This study aimed to identify the fungi associated with wilt disease complex of tomatillo in Sinaloa by the combination of phylogenetic analyses and morphological characterization, as well as to determine their pathogenicity and virulence on tomatillo seedlings.

Materials and methods

Sample collection

During several surveys carried out during 2016 and 2017 seasons, a total of 95 plants showing wilt and root rot symptoms were collected from 19 commercial tomatillo fields distributed in the main production area in northern Sinaloa (Ahome, El Fuerte, and Guasave municipalities), Mexico.

Isolation, purification, and conservation of fungi

For isolation, pieces of roots (5 ⋅ 5 mm) were taken from the margin between necrotic and healthy tissues. Their surface was sterilized by dipping in 2% sodium hypochlorite solution (NaOCl) for 1 min, rinsed two times with sterile distilled water and dried on sterilized paper. The pieces were placed in Petri plates with potato dextrose agar (PDA) (Difco, Detroit, MI, USA). The plates were incubated at 25 ºC for 3 days in darkness. Then, mycelial plugs (5 mm in diameter) from the edge of fungal colonies were aseptically transferred to fresh PDA. For isolates morphologically resembling Rhizoctonia and Macrophomina, pure cultures were obtained by transferring hyphal tips from the colony margin onto fresh PDA and incubated at 25 °C in the dark. For Fusarium-like colonies, single germinated conidia were removed and transferred to a new Petri plate containing PDA. Cultures of each isolate were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of Fuerte Valley (FAVF) at the Sinaloa Autonomous University (Juan Jose Rios, Sinaloa, Mexico).

Morphological characterization

A total of 37 isolates (15 of Rhizoctonia, 14 of Fusarium, five of Macrophomina, and three of Neocosmospora) were selected as representatives. Fusarium and Neocosmospora isolates were incubated at 25 °C with a 12-h photoperiod for 12 days on PDA and Carnation Leaf Agar (CLA) to examine the shape and size of macro, microconidia, and chlamydospores (Leslie and Summerell 2006) using a Leica TCS-SP5X confocal microscope (Leica, Germany). For Rhizoctonia isolates, young hyphae (5-days-old) of each isolate were stained with Safranin O + 3% KOH (Sneh et al. 1991) and the number of nuclei was determined for 20 cells. For Macrophomina isolates, slides containing fungal structures (mycelium and sclerotia) were stained with lactophenol cotton blue and observed under a light microscope.

DNA extraction, PCR amplification, and sequencing

Molecular identification was performed for 37 fungal isolates. Aerial mycelium from a 6-day-old culture was scraped from the medium using a sterile spatula and was placed in 2 mL microtubes. Total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the protocols provided by the manufacturer. DNA concentrations were quantified using a NanoDrop One (Thermo Fisher Scientific, Madison, WI, USA) and the samples were diluted to 100 ng µL− 1 for polymerase chain reaction (PCR).

For Rhizoctonia isolates (n = 15), the internal transcribed spacer (ITS) region of ribosomal DNA was amplified. For Fusarium (n = 14) and Neocosmospora (n = 03) isolates, part of the translation elongation factor 1-alpha (EF-1α) gene was amplified. For Macrophomina isolates (n = 05), the ITS region and part of EF-1α, β-tubulin (BT2), and actin (ACT) genes were amplified. PCR amplifications were conducted in a Bio-Rad CFX96 thermocycler (Bio-Rad Laboratories, Hercules, CA, USA), differently programmed for each primer set (Table 1). The PCR products were separated by electrophoresis in 1% agarose gels stained with ethidium bromide and the images were documented with the Gel Doc XR + System (Bio-Rad Laboratories, Hercules, CA, USA). The amplicons were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and both strands were sequenced by Macrogen (Macrogen Inc., Seoul, Korea) using the same primers that were used for amplification. All sequences generated in this study were deposited in GenBank (Table 2).

Table 1 Primers used in this study, with PCR conditions and references
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Table 2 GenBank accession numbers of DNA sequences of fungal isolates included in the phylogenetic analyses. Newly deposited sequences are shown in bold
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Phylogenetic analysis

To determine the identity of the 37 isolates to species level, phylogenetic analyses were conducted. The phylogeny was reconstructed by analyses from ITS sequences for Rhizoctonia, EF-1α sequences for Fusarium, ITS and EF-1α sequences for Neocosmospora, and ITS, EF-1α, BT2, and ACT sequences for Macrophomina. For the last two genera, phylogenetic reconstructions were performed using the concatenated sequences of the respective genes obtained with the SequenceMatrix (Vaidya et al. 2011). Forward and reverse sequences were assembled using the Staden Package (Staden et al. 1998). Multiple sequence alignments for each locus were independently performed using ClustalX v.1.83 (Thompson et al. 1997), using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and various alignment parameters (gap opening = 10, gap extension = 0.2, transition weight = 0.5, divergent delay sequences = 25%). The alignments were manually adjusted when necessary. The events of indels (gaps) were considered in the phylogenetic analysis (Young and Healy 2003). Nucleotide sequences of reference isolates for each genus obtained from GenBank were included in the analyses. The evolutionary history was inferred by using the Maximum Likelihood method and performed using the MEGA 7 (Kumar et al. 2016).

Pathogenicity and virulence tests

Pathogenicity tests involving fungal isolates of Rhizoctonia (n = 15), Fusarium (n = 14), Macrophomina (n = 05), and Neocosmospora (n = 03) were performed on tomatillo seedlings (cv. Gran Esmeralda) under greenhouse conditions. Tomatillo seeds were surface disinfested with 2% NaOCl solution for 1 min, rinsed two times with sterile distilled water and dried on sterilized paper. Seeds were germinated for four days on wet filter paper in glass Petri dishes at room temperature. Two germinated seeds were planted into 500 mL plastic pots containing 250 g of a sterilized substrate composed of sand + peat moss (1:3) and inoculated with four mycelial plugs (5 mm in diameter) removed from the margin of a 5-day-old PDA culture. Four non-colonized agar plugs were used on control plants. The pots were incubated under greenhouse conditions at 20–30 °C. Pots were irrigated when necessary to avoid dryness and fertilized with complex NPK solution after planting and every week. Three replicates (pots) arranged in a completely randomized design were used per each isolate. Twenty-five days after inoculation, plants were removed from pots, the roots were gently washed under running tap water and dried with paper towels. The virulence of each isolate was assessed by measuring disease severity using Correll et al. (1986) scale with some modifications, where: 0 = no discoloration; 1 = slight discoloration of the vascular tissue in roots; 2 = extensive discoloration of the vascular tissue in roots; 3 = slight discoloration of vascular tissue and cortex; 4 = extensive discoloration of vascular tissue and cortex; 5 = root completely necrotic or dead seedling. The entire experiment was done twice, obtaining similar results in both tests. Differences in virulence caused by soilborne fungi were determined by nonparametric analysis of variance (ANOVA) with Kruskal-Wallis test and rank average values were compared by Conover test.

Results

Fungal isolation

A total of 88 fungal isolates belonging to the genera Fusarium (n = 39), Rhizoctonia (n = 21), Macrophomina (n = 16), and Neocosmospora (n = 12) were obtained from diseased roots of tomatillo plants grown in 19 commercial fields in northern Sinaloa. Subsequently, 37 isolates were selected as representatives of the genera and geographical regions and were included in the morphological characterization, phylogenetic analysis, and pathogenicity tests.

Morphological characterization

Based on morphological features, 15 isolates were tentatively identified as Rhizoctonia solani, 11 belonging to Fusarium oxysporum species complex (FOSC), five as Macrophomina sp., three belonging to Fusarium solani species complex (FSSC), and three belonging to Fusarium fujikuroi species complex (FFSC).

Isolates belonging to FOSC exhibited cottony colonies with pigmentation varying from white to purple or violet on PDA. On CLA, macroconidia were falcate, hyaline, 3 to 4–septate and measuring 23.4–34.7 × 2.9–5.1 µm. On CLA, microconidia were hyaline, oval to ellipsoidal, aseptate or 1–septate, of 8.4–17.6 × 3.1–5.1 µm, and abundantly formed in false heads on short monophialides (Fig. 1).

Fig. 1
figure 1

Morphology of Fusarium oxysporum (a, d, g and j), F. nygamai (b, e, h and k), and Neocosmospora falciformis (c, f, i, l and m). a–c. Surface of colony growth on PDA after 7 days; d–f. Macroconidia; g–i. Microconidia; j–m. Monophialides. Scale bar = 10 µm

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Isolates belonging to FFSC exhibited cottony colonies with pigmentation that was white with a violet mass in the center on PDA. Macroconidia were falcate to almost straight, hyaline, 3–4-septate and measuring 20.5–52.7 × 3.1–4.9 µm. Microconidia were oval to elliptical, hyaline, aseptate or 1–septate, of 3.9–13.3 × 2.6–4.7 µm, and developed in false heads on short monophialides (Fig. 1).

Isolates belonging to FSSC showed cottony colonies with pigmentation varying from white to cream on PDA. On CLA, macroconidia were falcate, hyaline, with pointed apexes, with 3 to 4 septa, measuring 23.1–37.1 × 4.8–5.8 µm. On CLA, microconidia were oval, ellipsoid or reniform, hyaline, with rounded apexes, with 0 to 1 septate, measuring 7.9–21.1 × 2.9–5.8 µm, and arranged in false heads at the tips of long monophialides (Fig. 1).

All R. solani isolates were found to be multinucleate (5–10 nuclei per cell). On PDA, colonies were light brown to brown with mycelial growth of 2.4 cm day− 1. Hyphae showed a right-angle branching pattern, with a constriction at the base and adjacent septa. Colonies of Macrophomina isolates were olivaceous grey and with numerous dark oblong sclerotia on PDA.

Phylogenetic analyses

The phylogenetic analyses inferred under the Maximum Likelihood criterion provided sufficient information to distinguish Fusarium, Rhizoctonia, Neocosmospora, and Macrophomina species associated with tomatillo wilt in northern Sinaloa, Mexico. The alignment characteristics and statistics are summarized in Table 3.

Table 3 Characteristics of the data partitions used for phylogenetic analyses in this study
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The Fusarium isolates dataset included 23 taxa, including three outgroup taxa, Neocosmospora lichenicola NRRL 34123, N. falciformis NRRL 32542 and N. falciformis NRRL 32928. Maximum likelihood analysis using the EF-1α gene sequences resulted in three well-supported clades, corresponding to the Fusarium species described previously (Fig. 2). The first clade with 11 isolates clustered with Fusarium oxysporum [Agricultural Research Service Culture Collection, Philadelphia, USA (NRRL) 26406, 26381, and 38445], with 100% bootstrap support. The second clade with three isolates clustered with Fusarium nygamai [NRRL52708 and Fusarium Research Center, Pennsylvania State University, USA (FRC) M1374], with 99% bootstrap support.

Fig. 2
figure 2

Maximum Likelihood tree generated from sequence analysis of the EF-1α gene dataset of Fusarium species. Bootstrap support values for Maximum Likelihood are indicated at the nodes. The isolates in this study are in bold. Neocosmospora lichenichola strain NRRL 34123, N. falciformis strain NRRL 32542 and N. falciformis strain NRRL 32928 are used as outgroups. The scale bar indicates the number of expected changes per site

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In the phylogenetic tree generated with the sequences of the ITS region and the EF-1α gene of the Neocosmospora isolates, composed of 29 taxa (including 1 outgroup taxon, Geejayessia cicatricum NRRL 22316), the three isolates obtained in that study grouped with isolates of N. falciformis (NRRL32542, CBS 47567 and CBS 31873) with 85% bootstrap support (Fig. 3).

Fig. 3
figure 3

Maximum Likelihood tree generated from sequence analysis of the EF-1α gene and ITS region dataset of Neocosmospora species. Bootstrap support values for Maximum Likelihood are indicated at the nodes. The isolates in this study are in bold. Geejayessia cicatricum strain NRRL 22316 is used as outgroup. The scale bar indicates the number of expected changes per site

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The datasets used for phylogenetic analysis of the Rhizoctonia isolates consisted of 75 taxa for R. solani and 76 taxa for binucleate Rhizoctonia (including one outgroup taxon, Athelia rolfsii FSR-052). Phylogenetic analysis of the ITS region showed that all the isolates obtained in this study belong to the R. solani AG4-HGI anastomosis group (Fig. 4).

Fig. 4
figure 4

Maximum Likelihood tree generated from sequence analysis of the ITS region of Rhizoctonia solani AG4. Bootstrap support values for Maximum Likelihood are indicated at the nodes. The isolates in this study are in bold. Athelia rolfsii strain FSR-052 is used as outgroup. The scale bar indicates the number of expected changes per site

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For the phylogenetic analysis of Macrophomina isolates, the combined ITS, EF-1α, BT, and ACT datasets consisted of 15 taxa, including one outgroup taxon, Botryosphaeria dothidea CMW8000. The alignment contained 1883 characters, of which 309 corresponded with the EF-1α gene, 674 corresponded with the BT gene, 288 corresponded with the ACT gene, and 611 corresponded with the ITS region. The five Macrophomina isolates (FAVF147, FAVF148, FAVF149, FAVF150, and FAVF140) clustered with M. phaseolina [Culture Collection of Pedro Crous, housed at Utrecht, The Netherlands (CPC) 21416 and 21420, and Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) 20547, 26381, and 38445], with 99% bootstrap support (Fig. 5).

Fig. 5
figure 5

Maximum Likelihood tree generated from the combined ITS, EF-1α, BT, and ACT sequence alignment of Macrophomina species. Bootstrap support values for Maximum Likelihood are indicated at the nodes. The isolates in this study are in bold. Botryosphaeria dothidea strain CMW8000 is used as outgroup. The scale bar indicates the number of expected changes per site

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Distribution of fungal species

Rhizoctonia solani AG 4–HGI was the most frequently identified species (40.5%) followed by F. oxysporum (29.8%), M. phaseolina (13.5%), N. falciformis (8.1%), and F. nygamai (8.1%) from the 37 fungal isolates obtained from tomatillo symptomatic samples. The distribution of fungal species varied among the populations obtained from tomatillo fields in the three municipalities located in northern Sinaloa, Mexico. Fusarium oxysporum, N. falciformis, and M. phaseolina were found in tomatillo fields from the three municipalities. Fusarium nygamai was only recorded in the Ahome and El Fuerte populations, whereas, R. solani was only identified in the Ahome and Guasave populations. The Ahome population had all fungal species identified in this study (Fig. 6).

Fig. 6
figure 6

Collection sites of fungal species associated with tomatillo wilt in three municipalities distributed in the state of Sinaloa, Mexico. Circles represent association frequency of each fungal species in each population sampled, “v” is the number of tomatillo fields sampled in each population and “n” is the number of isolates analyzed in each population

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Pathogenicity tests

All isolates of Fusarium, Rhizoctonia, Macrophomina, and Neocosmospora were pathogenic in tomatillo seedlings. Inoculated seedlings developed symptoms of root rot, poor growth, discoloration of vascular tissues, wilting, and death of plants whereas control plants remained symptomless. Fungal colonies were re-isolated from all symptomatic plants and were found to be morphologically similar to the original isolates inoculated on tomatillo seedlings, thus fulfilling Koch’s postulates. Additionally, there were significant differences (H = 72.04, P ≤ 0.05) in virulence between the isolates of each species, although the disease was evident in all the fungal species analyzed, only 17 isolates were different from the control (Fig. 7). Rhizoctonia solani isolates were the most virulent (H = 36.29, P ≤ 0.05), followed by the rest of the fungal species (Fig. 8). The pathogenicity data of F. nygamai and N. falcifomis were verified using the Mann-Whitney-Wilcoxon test and in both cases their virulence with respect to the control was identified (P ≤ 0.05).

Fig. 7
figure 7

Mean disease severity caused by 37 fungal isolates associated with tomatillo wilt in northern Sinaloa, Mexico, 30 days after inoculation with mycelium colonized agar plugs onto roots of Physalis ixocarpa seedlings. Isolates with the same letter do not differ significantly (P ≤ 0.05)

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Fig. 8
figure 8

Mean disease severity caused by five fungal species associated with tomatillo wilt in northern Sinaloa, Mexico, 30 days after inoculation with mycelium colonized agar plugs onto roots of Physalis ixocarpa seedlings. Bars above columns are the standard errors of the means. Fungal species with the same letter do not differ significantly (P ≤ 0.05)

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Discussion

Based on phylogenetic analysis of the ITS region from 15 Rhizoctonia isolates, it was determined that all of them belonged to R. solani AG 4-HGI. These isolates were obtained from tomatillo fields located in the municipalities of Ahome and Guasave, Sinaloa. Rhizoctonia solani is a soil borne fungus with a wide range of hosts and cosmopolitan distribution (Koike et al. 2007). In Mexico, the presence of R. solani on tomatillo has been reported, however, its anastomosis group has not been determined (Apodaca-Sánchez et al. 2008). Predominance of R. solani AG 4-HGI isolates associated with tomatillo plants in Sinaloa agrees with previous reports indicating a wide distribution of this anastomosis group in Mexico. Also, the anastomosis group AG-4 was identified as the most common in the rhizosphere of grapevine (Vitis vinifera) on the coast of Hermosillo in the state of Sonora, Mexico (Meza-Moller et al. 2007, 2011). Similarly, Montero-Tavera et al. (2013) determined that anastomosis group AG-4 was widely distributed in fields of chili pepper (Capsicum annuum) in the Mexican states of Zacatecas, San Luis Potosí, Durango, Guanajuato, Chihuahua, Colima, and Querétaro. Furthermore, Elias-Medina et al. (1997) reported the presence of this anastomosis group in plants of chili pepper and common bean (Phaseolus vulgaris) in the State of Mexico and San Luis Potosí, respectively. In contrast, Virgen-Calleros et al. (2000) found that anastomosis group AG-4 was the second most frequently isolated after anastomosis group AG-3 in potato fields (Solanum tuberosum) in Guanajuato, Mexico.

In the current study, 14 Fusarium isolates were identified through phylogenetic analysis of EF-1α sequences data, determining that 11 isolates belonged to F. oxysporum and three isolates to F. nygamai. Regarding F. oxysporum, this species has been previously reported causing wilt symptoms on tomatillo plants in Sinaloa (Apodaca-Sánchez et al. 2008) and State of Mexico (Gómez-Camacho et al. 2006). Similarly, this fungus has been reported as the causal agent of vascular wilt, which is the most devastating disease that affects cape gooseberry (Physalis peruviana) in Colombia (Urrea et al. 2011; Enciso-Rodríguez et al. 2013; Osorio-Guarín et al. 2016). Soto et al. (1998) found differences in susceptibility of 95 genotypes of tomatillo in Mexico due to natural infection by Fusarium sp. under field conditions. However, further studies should evaluate the response of tomatillo genotypes produced in Mexico to the infection by each fungal species associated with symptoms of the disease, in order to identify the levels of resistance and tolerance to these pathogens.

On the other hand, F. nygamai has been mainly found in hosts of the Poaceae, including Pennisetum typhoides, Sorghum bicolor, Zea mays and Saccharum officinarum. Concerning Solanaceous hosts, F. nygamai only has been reported in tobacco (Nicotiana tabacum) (Farr and Rossman 2019). In Mexico, F. nygamai was associated with root necrosis on sugarcane (Saccharum officinarum) in the state of Morelos (Martínez-Fernández et al. 2015). Likewise, Leyva-Madrigal et al. (2015) reported that F. nygamai was the most prevalent and one of the most aggressive species causing root rot on maize in northern Sinaloa. In the current study, F. nygamai isolates were significantly less virulent and less abundant on tomatillo plants as compared with F. oxysporum isolates. The magnitude of change in severity shown by F. nygamai and N. falcifomis did not allow the identification of their pathogenicity with respect to the control in the analysis carried out using Kruskal-Wallis test, so only for the purpose of providing evidence for the pathogenic capacity of these species the Mann-Whitney-Wilcoxon test was performed. To the best of our knowledge, this is the first report of F. nygamai isolates associated with wilt symptoms in tomatillo in Mexico and worldwide.

Based on EF-1α sequences, three Neocosmospora isolates included in this study were identified as N. falciformis (syn. F. falciforme). This fungus is included in the Fusarium solani species complex which has more than 15 phylogenetic species (Sandoval-Denis and Crous 2018). Recently, N. falciformis has been reported causing root rot on lima bean (Phaseolus lunatus) in Brazil (Sousa et al. 2017), basal rot on onions (Allium cepa) (Tirado-Ramírez et al. 2018), root rot on watermelon (Citrullus lanatus) (Rentería-Martínez et al. 2018) and tomato wilt (Solanum lycopersicum) (Vega-Gutiérrez et al. 2019a) in Mexico. It has also been reported causing root and stem rot on papaya in Mexico (Vega-Gutiérrez et al. 2019b) and India (Gupta et al. 2019). However, this is the first report of N. falciformis infecting P. ixocarpa in Mexico and worldwide.

The phylogenetic analysis generated with sequence data (ITS, EF-1α, BT, and ACT) indicated that all Macrophomina isolates tested in this work belonged to M. phaseolina. The genus Macrophomina belongs to the family Botryosphaeriaceae and it has four phylogenetic species: M. phaseolina, M. pseudophaseolina, M. euphorbiicola, and M. vaccinii (Machado et al. 2019; Zhao et al. 2019). Previous studies showed that M. phaseolina has been associated with Physalis peruviana (Shivas 1989) in Australia and Pakistan (Ahmad et al. 1997) and on Physalis minima in Australia (Fuhlbohm et al. 2012). In Mexico, M. phaseolina has been detected on a wide range of host plants from different families (Muñoz-Cabañas et al. 2005) and with a wide distribution in arid, tropical and subtropical regions (Mayek-Pérez et al. 2001), but has not been previously reported on plants of the genus Physalis. Thus, this is the first report of M. phaseolina infecting P. ixocarpa in Mexico and worldwide.

Our results of pathogenicity tests revealed that isolates of R. solani were more virulent in tomatillo plants as compared with F. oxysporum, M. phaseolina, N. falciformis, and F. nygamai isolates. In a similar study, Güney and Güldür (2018) found no significative differences in severity of foliar symptoms and root rot induced by R. solani, F. oxysporum, M. phaseolina and N. solani on artificially inoculated chili pepper plants. Other authors like Datnoff and Sinclair (1988) found that severity in root rot of soybean (Glycine max) plants inoculated with F. oxysporum and R. solani was not statistically different. Nevertheless, their results demonstrated an additive interaction with severity of the disease increasing with the presence of both pathogens. Finally, Navarrete-Maya et al. (2009) stated that the virulence of R. solani was less severe compared to that of Fusarium spp. in common bean plants under field conditions, but in a controlled environment, the virulence of R. solani was more severe.

This work provides new information about identification and pathogenicity of soilborne fungi associated with tomatillo wilt in northern Sinaloa, Mexico. It also reported for the first time the presence of M. phaseolina, N. falciformis and F. nygamai isolates associated with this disease. These findings are part of further epidemiological studies that will be used to establish effective management strategies of tomatillo wilt in commercial fields of Mexico.