Alfalfa (Medicago sativa) is a perennial crop that accounts for 27% of national forage production. In Mexico, the area sown to alfalfa in 2018 was a bit more than 387,000 hectares with an average yield of green forage of 58.7 t ha^-1 year^-1 (^{SIAP, 2018}). The states with the highest alfalfa production at the national level are Chihuahua, Guanajuato, Hidalgo, Baja California, Sonora, Durango, Coahuila and Puebla, which together account for 70% of national production (^{Lara and Jurado, 2014}). Alfalfa is an important crop in north-central Mexico, where the states of Aguascalientes, Durango (Los Llanos) and Zacatecas are located. Large variable-sized spots of dead alfalfa plants have been observed in most commercial plots, but there is little or no information about the organisms that cause crown rot or the extent of losses caused by the disease. Alfalfa crown rot is spread throughout the world. Several Fusarium species are among the fungi most commonly isolated from necrotic crowns and roots of alfalfa plants (^{Uddin and Knous, 1991}); in Sudan, ^{Ao et al. (2018)} mention that Fusarium, Rhizoctonia and some Pythium species are present in alfalfa plots. On the other hand, in Mexico, ^{Chew (2000)} noted there was a high incidence of crown rot in alfalfa plots in the Comarca Lagunera (region of lagoons). In the Mexican state of Guanajuato, F. incarnatum was reported as being the causal agent of alfalfa root rot (^{Esteban-Santiago et al., 2016}). ^{Velásquez-Valle (2001)} reported the presence of galls on the roots of alfalfa plants collected in Aguascalientes. Other studies have reported the presence of bulb nematodes (Ditylenchus dipsaci) on alfalfa leaf samples collected in the state of Jalisco (^{Rosas-Hernández et al., 2017}). Alfalfa productivity in this region could increase by implementing phytosanitary measures to reduce the crown rot problem. However, these measures must be solidly based on knowledge of the organisms that could be the potential causes of the disease. The objective of this study was to identify the genera of microorganisms associated with alfalfa plants presenting crown rot symptoms in Aguascalientes, Durango and Zacatecas, Mexico.

Between January and August 2017, samples of alfalfa roots with crown rot symptoms and of the soil surrounding the plants were collected from 30 commercial plots, of which 50, 30 and 20% were in Zacatecas, Aguascalientes and Durango, respectively. Three stunted plants with a few yellow leaves, most of them with wilt symptoms, were randomly collected from each plot. The plants were extracted with as many of the roots as possible, and a subsample of the soil surrounding each plant was taken; this soil was mixed together to obtain a representative compound sample. The crown of each plant was rinsed with tap water and tissue was taken from its center. The tissue was then disinfected and cultivated in Petri dishes containing a potato-dextrose-agar (PDA) culture medium (^{Velásquez-Valle et al., 2001}). The Petri dishes were incubated at 28 ± 1 °C in a bacteriological oven (Felisa, Mod. FE 131) for three or four days. The genera of the resulting colonies of fungi or oomycetes were identified using the taxonomical keys of ^{Barnett (1967)} and ^{Watanabe (1994)}. To extract thread-like nematodes, each soil sample was homogenized and then a 50-g subsample of each was taken and placed in a Baermann funnel, according to the methodology proposed by ^{Cepeda (1995)}. The genera of the recovered nematodes were identified using the taxonomic keys and information provided by ^{Mai and Mullin (1996)}, ^{Kanzaki and Giblin-Davis (2012)}) and ^{Castillo et al. (2012)}). The roots were examined to detect galls caused by the Meloidogyne nematode.

The results showed there were five genera of fungi or oomycetes on the root samples that are potentially phytopathogenic to the crop (Fusarium, Rhizoctonia, Verticillium, Pythium and Phytophthora), while the results of soil samples showed 13 genera of phytoparasite, predator and free-living nematodes (Aphelenchus, Aphelenchoides, Criconemoides, Ditylenchus, Dorylaimus, Helicotylenchus, Mononchus, Pratylenchus, Psilenchus, Rhabditis, Trichodorus, Tylenchus and Xiphinema). The most frequently identified nematodes were Aphelenchus, Rhabditis and Dorylaimus (96.7, 73.3 and 70%, respectively). In contrast, Criconemoides, Trichodorus and Xiphinema were identified the least frequently (3.3% in each case) (Table 1).

It should be noted that Ditylenchus was present in approximately 57% of the alfalfa plots sampled in the three states, it is important to highlight a study conducted by ^{Boelter et al. (1985)}, who indicated the association of D. dipsaci with the death in winter of alfalfa plants and reduced forage yield. Thus, it could be that the damage caused by the nematode in this region of Mexico is underestimated or mistaken for the effect of low winter temperatures. On the other hand, we should highlight that the nematode was not present in samplings conducted by ^{Chew (2000)} in alfalfa plots in the Comarca Lagunera. ^{Williams-Woodward and Gray (1999)}, and ^{Milano de Tomasel and McIntyre (2001)} also pointed out that D. dipsaci infestations frequently occur in conjunction with Aphelenchoides ritzemabosi, both in the soil and on alfalfa foliage. In the present study, we detected the simultaneous presence of Aphelenchoides and Ditylenchus in soil samples taken from two alfalfa plots in southeastern Zacatecas. Aphelenchoides was also found in two alfalfa plots in Durango, but not Ditylenchus.

According to ^{Castillo et al. (2012)}, Pratylenchus penetrans is distributed all over the Americas, where it parasitizes more than 350 hosts, including alfalfa. The results of this study show that specimens of the Pratylenchus genus were found in 50% of the alfalfa plots sampled in the three states. According to ^{Chew (2000)}, this nematode is present in alfalfa plots in Coahuila and Durango. ^{Mauza and Webster (1982)} demonstrated a synergistic interaction between P. penetrans and Fusarium oxysporum populations in alfalfa seedlings. Although F. solani was not pathogenic to alfalfa seedlings, it reduced P. penetrans populations. It is important to point out that in this study the presence of Fusarium in alfalfa plants’ crown tissue coincided with the presence of Pratylenchus in the soil samples, though this information can be confirmed later by extracting nematodes from the crown tissue.

The Aphelenchus genus includes species considered to be biological control agents of fungal microorganisms and facultative phytopathogenic species, although severe damage has not been reported on major agricultural species (^{Kanzaki and Giblin-Davis, 2012}). Aphelenchus avenae is widely distributed on decaying rhizomes, tubers, bulbs and roots. It was found on lesions caused by Pythium arrhenomanes on maize roots (Zea mays) but not on healthy roots (^{Mai and Mullin, 1996}). The high incidence of this nematode (96.7%) found in this study is probably due to the decaying tissues in the plant’s crown.

In one third of soil samples, specimens belonging to Tylenchus were found. According to ^{Thorne (1961)}, members of the Tylenchus family are among the nematodes most frequently found in the rhizosphere; however, except for T. costatus, they have not been reported as phytoparasites.

A Xiphinema specimen was identified in an alfalfa plot in Zacatecas; it had been previously reported (^{Chew, 2000}) on alfalfa crops in the states of Coahuila and Durango. ^{Wojtowicz et al. (1982)} also reported the incidence of this genus on alfalfa in the state of Pennsylvania, USA. Trichodorus was detected in an alfalfa plot in Aguascalientes. It is important to mention that ^{Thorne (1961)} had indicated that alfalfa was not a good host of this nematode.

No galls caused by nematodes such as Meloidogyne were detected, and this result is in agreement with that obtained by ^{Chew (2000)} in alfalfa plots in the Comarca Lagunera. However, a previous report mentioned the presence of galls caused by this nematode in an alfalfa plot in Aguascalientes (^{Velásquez-Valle, 2001}).

The alfalfa plants collected had scant foliage, most frequently a dull green to yellowish in color, and smaller in size than non-affected plants; other symptoms such as stunting and necrosis were observed as well. The crown of these plants had brown-to-black lesions frequently extending towards the root’s vascular tissues, according to the syndrome described by ^{Uddin and Knous (1991)}.

Based on these symptoms, five genera of microorganisms were isolated and identified: Fusarium, Rhizoctonia and Verticillium fungi, as well as Phytophthora and Pythium oomycetes (Table 2).

It is worth mentioning the high incidence of Fusarium and Rhizoctonia in alfalfa plots in each state, and consequently, at the regional level; these two fungi are associated with crown rot (Table 2). A group of Fusarium species has been associated with the disease: F. incarnatum, F. solani, F. roseum, F. oxysporum, among others (^{McKenzie and Davidson, 1975}; ^{Mauza and Webster, 1982}; ^{Esteban-Santiago et al., 2016}). We need to keep in mind that not all the Fusarium species are pathogenic to alfalfa (^{Mauza and Webster, 1982}), so further identification and pathogenicity research needs to be done in order to define the role they play in alfalfa crown rot. According to ^{Chew (2000)}, Fusarium sp. was the fungus most frequently found on alfalfa plants with crown rot symptoms in the Comarca Lagunera, which is in agreement with the results of this study.

Rhizoctonia solani has been reported to be the causal agent of the damping-off of alfalfa seedlings and of lesions at the base of alfalfa sprouts (^{Vincelli and Herr, 1992}; ^{Fowler et al., 1999}). In the Comarca Lagunera, ^{Chew (2000)} reported that after Fusarium, Rhizoctonia solani was the fungus found most frequently on alfalfa plants with crown rot.

According to ^{Larkin et al. (1995)}, some alfalfa root microorganisms, such as Phytophthora and Pythium, have been associated with limited development and establishment of alfalfa populations. Phytophthora was identified in two alfalfa plots in the municipalities of Calera and Luis Moya, Zacatecas. Some Phytophthora species have been classified as pathogenic to alfalfa. For example, alfalfa roots infected by P. megasperma reduced the development rate in adult plants, and severe infections caused symptoms such as yellow foliage, wilting and premature defoliation (^{Marks and Mitchell, 1971}). On the other hand, the incidence of Pythium in the region was 53.3% (Table 2). A similar situation was reported in Sudan (^{Ao et al., 2018}), where the combined incidence of four Pythium species reached 44%.

In conclusion, 13 genera of nematodes were identified (Aphelenchus, Aphelenchoides, Criconemoides, Ditylenchus, Dorylaimus, Helicotylenchus, Mononchus, Pratylenchus, Psilenchus, Rhabditis, Trichodorus, Tylenchus and Xiphinema) in the rhizosphere of alfalfa plants with crown rot symptoms; three genera of fungi (Fusarium, Rhizoctonia, Verticillium) and two of oomycetes (Phytophthora and Pythium) were associated with crown rot on alfalfa plants.