Nematophagous Fungi Occurrence: Prediction Using Bioclimatic Variables

Abstract

This study aimed to identify bioclimatic variables that favour the occurrence of three fungal species of the genus Arthrobotrys. For this purpose, 122 samples were collected from agricultural soils, 41 of which were positive for nematophagous fungi. In total, 13 pure Arthrobotrys spp. cultures tested positive for pathogenicity to entomopathogenic nematodes and were identified at the species level based on their morphology and morphometry. The environmental and bioclimatic characteristics of positive sampling sites were evaluated using the maximum entropy algorithm, with 22 bioclimatic variables as predictors; among them, the main variables that promoted the occurrence of Arthrobotrys spp. were moisture regime (35.1%), precipitation of warmest quarter (21.3%), and altitude (20.5%). The total surface area with these conditions was 109,568 ha. In Guanajuato, Mexico, conditions favour the occurrence of nematophagous fungi. The bioclimatic variables that increased the incidence of the genus Arthrobotrys were moisture regime, precipitation of the warmest quarter, and altitude. The municipalities in Guanajuato of Abasolo (001), Irapuato (017), Jaral del progreso (018), León (020), Pueblo Nuevo (024), Salamanca (027), and Valle de Santiago (042) encompass regions conducive to finding nematophagous fungi.

1. Introduction

The distribution, abundance, and activity of microorganisms have mainly been demonstrated in temperate habitats with soil organic matter whose content depends on environmental conditions [1,2]. Soil supports most fungal biodiversity; however, estimates of the number of soil species are inconsistent [3,4,5]. Soil fungi are ecologically relevant because of their wide distribution and functions and have been isolated from almost all environmental niches [6,7].

According to Tedersoo, [8] the key indicators that favour fungal abundance in the environment are climatic factors, the water table, and soil porosity and texture. Additionally, fungi are more frequently isolated from silty loam soils than from clay soils [9].

More than 700 fungal species feed on nematodes [10]. These microorganisms that trap and feed on nematodes are known as nematophagous fungi [11,12]. Plant-parasitic or root-knot nematodes cause substantial economic losses [13] and limit the production of various agricultural crops, accounting for 14% of annual yield losses in total global production [14]. Notably, the activity of nematophagous fungi is affected by soil pH, moisture, and temperature [15].

The earliest description of a nematophagous fungus, Arthrobotrys superba, dates back to 1839; however, its predatory capacity was not recognized until 1888. The genus Arthrobotrys is characterized by hyaline conidiophores that produce conidia asynchronously on short denticles arising from swollen conidiogenous heads [16]. The conidia are typically subhyaline, obovoid to clavate, and septate. Notably, species within this genus exhibit a diverse array of sophisticated nematode capture devices, including constricting rings, non-constricting rings, adhesive networks, adhesive hyphae or knobs (buttons), stephanocysts, spiny balls, and acanthocytes, all of which differentiate from vegetative mycelium [17].

In different ecological niches, fungal populations vary due to many factors, such as nutrient concentration, growth limitations, and altitude [8,18]. Additionally, results from biodiversity and climate research have demonstrated the negative effects of climate change on beneficial and pathogenic microorganisms [19]. In this context, increasing the efficiency of detecting soil microorganisms is necessary and requires applying methods to characterize bioclimatic variables and assess the climate impact on populations and agricultural production.

The potential (current, historical, and future) distribution of microorganisms such as nematophagous fungi has been predicted using bioclimatic models [20]. Guisan and Zimmerman [21] and López-Rocha [20] have created species distribution models based on ecological and bioclimatic data for understanding the occurrence of microorganisms in different ecological niches by using the principle of maximum entropy. The maximum entropy algorithm (MaxEnt) calculates the probability of occurrence of areas suitable for a species from specific presence data without requiring absence data, generating accurate predictions [22,23].

However, according to distribution studies, the environmental suitability for or the probability of occurrence of nematophagous fungi remains low. Moreover, according to a review of the literature, the bioclimatic variables of nematophagous fungi have not been investigated. Therefore, this study aimed to determine the bioclimatic variables that condition the distribution of three fungal species of the genus Arthrobotrys.

2. Materials and Methods

2.1. Study Area and Period

This study was conducted in two stages in the state of Guanajuato, where the temperature ranges from 7.5 to 28.8 °C, averaging 18 °C, and the relative air humidity ranges from 29% to 71% [24]. In the first stage, from August 2021 to October 2022, soil samples were collected from agricultural fields by using the five-point sampling method [25,26] and stored in coolers at 25 ± 1 °C. In the second stage, nematophagous fungi were isolated and identified before their pathogenicity was tested, their predatory capacity was determined, and the environmental suitability model was applied at the Parasitology and Biological Control Laboratory (Laboratorio de Parasitología y Control Biológico; LPCB), Division of Life Sciences, University of Guanajuato (Universidad de Guanajuato—UG).

2.2. Isolation and Identification of Nematophagous Fungi

Nematophagous fungi were isolated by placing soil samples along with the roots of the collected plants on filter paper in Petri dishes sterilized with 70% ethanol. The Petri dishes were inoculated using the soil sprinkling technique [27] under field conditions of permanent moisture and darkness at 25 ± 1 °C.

Each Petri dish was placed under a stereoscopic microscope (candelabra observation) for the identification of nematophagous fungi, which were picked and seeded in a Petri dish with water agar. This procedure was repeated as many times as necessary to obtain a pure culture. When the pure culture was detected, monosporic cultures were prepared from a concentration of 1 × 10⁶ conidia inoculated in water agar by using a cell spreader until homogeneous cultures were obtained. Nematophagous fungi were identified at the species level based on the morphology and morphometry of their structures, which were observed under a compound microscope, according to the species identification keys of Baral et al. [28]. Cooke et al. [29], and Swe et al. [30].

2.3. Pathogenicity of Nematophagous Fungi to Entomopathogenic Nematode Larvae

The pathogenicity of nematophagous fungi was assessed by performing a qualitative assay using pure monosporic cultures of isolates and larvae of entomopathogenic nematodes from the LPCB collection, UG [31]. First, nematophagous fungi isolates were sub-cultured in Petri dishes with corn flour agar and grown for 21 days after inoculation. Subsequently, approximately 100 infective juveniles of entomopathogenic nematodes were added to each Petri dish and incubated for 7 days after inoculation at 26 ± 1 °C in the dark [32]. These infective juveniles, belonging to the genus Heterorhabditis, were originally isolated from soils used for livestock production (dairy cows) and propagated in the laboratory with larvae of the insect Galleria mellonella. For each nematophagous fungi isolate, three replicates and one negative control with water agar were prepared with entomopathogenic nematodes in all treatments. The isolates that damaged nematode larvae were deemed pathogenic, and the nematophagous fungi were re-isolated to confirm that the organisms were pathogenic in accordance with Koch’s postulates [33].

2.4. Environmental Suitability Model

Herein, the database of the occurrence of nematophagous fungi and MaxEnt software version 3.3.3 [34] were used to develop an environmental suitability model. For this purpose, 22 climatic variables were used as predictors (

Table 1. Environmental and bioclimatic variables used to estimate the environmental suitability of the genus Arthrobotrys and the species Arthrobotrys thaumasia, Arthrobotrys musiformis, and Arthrobotrys oligospora in Guanajuato, Mexico.

Code	Variable	Unit
Bio1	Annual mean temperature	°C
Bio2	Mean diurnal range	°C
Bio3	Isothermality	Dimensionless
Bio4	Temperature seasonality	CV
Bio5	Maximum temperature of warmest month	°C
Bio6	Minimum temperature of coldest month	°C
Bio7	Temperature annual range	°C
Bio8	Mean temperature of wettest quarter	°C
Bio9	Mean temperature of driest quarter	°C
Bio10	Mean temperature of warmest quarter	°C
Bio11	Mean temperature of coldest quarter	°C
Bio12	Annual precipitation	mm
Bio13	Precipitation of wettest month	mm
Bio14	Precipitation of driest month	mm
Bio15	Precipitation seasonality	CV
Bio16	Precipitation of wettest quarter	mm
Bio17	Precipitation of driest quarter	mm
Bio18	Precipitation of warmest quarter	mm
Bio19	Precipitation of coldest quarter	mm
Bio20	Altitude	m
Bio21	Moisture regime	°
Bio22	Vegetation	23 types

), 19 of which were bioclimatic variables with a spatial resolution of 0.5′ retrieved from the WorldClim database(www.worldclim.org (accessed on 18 September 2023). Digital elevation model data (with 90 m resolution) were extracted from the Consultative Group for International Agricultural Research–Consortium for Spatial Information (CGIAR-CSI; http://srtm.csi.cgiar.org, formerly CGIAR). Land use and vegetation vector layers were from the Mexican Commission for the Knowledge and Use of Biodiversity (Comisión Nacional para el Conocimiento y Uso de la Biodiversidad—CONABIO) [35] and the soil moisture regime [36].

3. Results and Discussion

3.1. Isolation, Identification, and Pathogenicity of Nematophagous Fungi

Of the 122 samples collected, 41 tested positive. In these 41 samples, three species were identified: Arthrobotrys oligospora, Arthrobotrys musiformis, and Arthrobotrys thaumasia. In addition, 13 nematophagous fungi of the genus Arthrobotrys were isolated, all of which infected at least one entomopathogenic nematode larva (Figure 1 and Figure 2). The pathogenic mechanisms of prey organisms were constricting rings and adhesive networks and traps (

Table 2. Identification and pathogenicity of nematophagous fungi of soils from Guanajuato, Mexico.

Sample	Municipality	Species	Pathogenicity Test
S00-AO	Valle de Santiago	Arthrobotrys oligospora	+
S02-AM	Salamanca	Arthrobotrys musiformis	+
S03-AT	Valle de Santiago	A. musiformis	+
S08-AM	Huanimaro	A. musiformis	+
S09-AO	Jaral del progreso	A. oligospora	+
S10-AO	Valle de Santiago	A. oligospora	+
S13-AO	Valle de Santiago	A. oligospora	+
S17-AT	Salamanca	Arthrobotrys thaumasia	+
S20-AM	Pénjamo	A. musiformis	+
S21-AT	Valle de Santiago	A. musiformis	+
S27-AM	Valle de Santiago	A. musiformis	+
S28-AT	Valle de Santiago	A. thaumasia	+
S29-AM	Valle de Santiago	A. oligospora	+

S = soil; +: pathogenic organism.

).

3.2. Evaluation of the Environmental Suitability Model

Of the 22 bioclimatic variables used as predictors in this study, three were the most crucial for determining the environmental suitability of the genus Arthrobotrys: moisture regime (35.1%), precipitation of warmest quarter (21.3%), and altitude (20.7%) (

Table 3. Percentage contribution of the main bioclimatic variables to the environmental suitability model for the genus Arthrobotrys.

Variable	Contribution (%)
	Arthrobotrys
Moisture regime (Bio 21)	35.1
Precipitation of warmest quarter (Bio18)	21.3
Altitude (Bio 20)	20.7
Temperature seasonality (Bio4)	9.9
Precipitation of driest month (Bio14)	2.8
Precipitation seasonality (Bio15)	2.6
Minimum temperature of coldest month (Bio 6)	2.4

). The minimum temperature of the coldest month, temperature seasonality, precipitation of the driest month, and precipitation seasonality variables were not significant. To the best of our knowledge, this study is the first to determine the bioclimatic variables of fungal species of the genus Arthrobotrys using an environmental suitability model. Gortari et al. [37] demonstrated that for the species diversity of nematophagous fungi from different ecosystems, some species were observed to be geographically restricted. Notably, research on nematophagous fungi of the genus Arthrobotrys in the context of agriculture is necessary. Peraza-Padilla et al. [26] demonstrated that most nematophagous fungi isolates have been identified in regions with high precipitation and relative air humidity (70%).

Additionally, the probability of occurrence of Arthrobotrys spp. increased (65%) when the moisture regime (soil moisture retention capacity > 50%) [38] surpassed 250 days, characteristic of an ustic moisture regime (180–270 days of humidity). Conversely, the probability of occurrence decreased to 0% when the number of days of humidity was >150 (Figure 3). According to Larsen et al. [39], fungi must adapt to unfavourable ecological niches, exposed to different regimes. Gray [40] observed that the occurrence of nematophagous fungi was affected by the percentage of humidity and the pH; Akhtar [41] and Jaffe [42] have demonstrated that the occurrence of nematophagous fungi is associated with abiotic factors; the two studies demonstrated consistent results showing that abiotic conditions determined the occurrence of nematophagous fungi in soils.

The probability of occurrence of Arthrobotrys spp. increased (90%) when the precipitation of the warmest quarter (May–August) ranged from 160 to 180 mm and decreased (to 0%) when the precipitation increased to >400 mm (Figure 4). This variable was associated with an increased occurrence of fungi of this genus. Peraza-Padilla et al. [26] demonstrated that high precipitation of approximately 3525 mm/year helps isolate fungi of the genus Arthrobotrys. These findings are not generalizable to all species and contradict those of this study. However, the findings of this study support those of García et al. [43], who found that the probability of occurrence of Fusarium oxysporum increased (68%) when the precipitation ranged from 150 to 180 mm.

For altitude, the probability of occurrence increased (90%) from 1750 m above sea level (masl), and in the state of Guanajuato, the probability decreased (to 0%) at altitudes lower than 700 masl (Figure 5). Peraza-Padilla et al. [26] showed that fungal species of the genus Arhrobotrys can be isolated at 42 masl; this variable may be notable because fungi proliferate at this altitude (42 masl). Nevertheless, these results should be confirmed with other similar or biological studies in the laboratory and the field because they are contradictory.

The environmental suitability model of Arthrobotrys spp. (Figure 6) showed above-average values of probability of occurrence (0.69–1.0) for seven municipalities: L Abasolo, (001) 8127 ha; Irapuato (017), 18,968 ha; Jaral del Progreso (018), 4514 ha; León (020), 5284 ha; Pueblo Nuevo (024), 5991 ha; Salamanca (027), 16,516 ha; and Valle de Santiago (042), 50,165 ha. The typical soils of these areas are pellic vertisols and haplic phaeozem soils, and precipitation in the driest month is <40 mm. The relationship between total precipitation and mean temperature is <43.2, suggesting that intense rainfall patterns have a stronger effect on the probability of occurrence than temperature and winter rainfall ranging from 5% to 10% of the total annual precipitation. The climate is classified as semi-warm subhumid group C: average annual temperature, >18 °C; temperature of the coldest month, <18 °C; and temperature of warmest month, >22 °C. The moisture regime is ustic (ranging from 180 to 270 days of humidity). In this study, the probability of occurrence of Arthrobotrys was high, mainly in areas of annual irrigation agriculture, annual rainfed agriculture, induced grassland, and secondary shrubby vegetation of low deciduous forest. These results are consistent with those of Peraza-Padilla et al. [26], Soto et al. [44], and Sánchez et al. [45], who demonstrated that nematophagous fungi thrived in various types of substrates, production systems, geographical areas, humus-rich soils, and forage grasses and that the quantity and quality of root exudates from mature plants had a positive effect on microbiological diversity.

4. Conclusions

In Guanajuato, Mexico, the conditions favour the occurrence of nematophagous fungi. The bioclimatic variables that promote the incidence of the genus Arthrobotrys are the moisture regime, precipitation of warmest quarter, and altitude.

The municipalities of Abasolo (001), Irapuato (017), Jaral del progreso (018), León (020), Pueblo Nuevo (024), Salamanca (027), and Valle de Santiago (042) are regions conducive to finding nematophagous fungi.