Assessing the predatory activity of Arthrobotrys oligosporus strain C-2197 as biocontrol of the root-knot nematode Meloidogyne spp.
Adela Quevedo1, Marcos Vera-Morales1, Fernando Espinoza-Lozano1, Rafael F. Castañeda-Ruiz3, Daynet Sosa1,2, Freddy Magdama1,2*
Available from: http://dx.doi.org/10.21931/RB/2021.06.01.22
The root-knot nematode, Meloidogyne spp., is an endoparasite that infects plants' root system and causes yield losses in several important crops. Meloidogyne is one of the most devastating pests, so searching for effective biological agents is needed to mitigate its damage. In this study, the predatory activity of Arthrobotrys oligosporus Fresen strain C-2197, obtained from a tropical dry forest of Ecuador, was evaluated as a biocontrol alternative for root-knot caused by Meloidogyne spp. Our results showed that A. oligosporus C-2197 has predatory activity against juvenile nematodes, 72.31%, and 79% efficacy, for in vitro and greenhouse conditions. Besides, the studied strain showed growth-promoting activity, increasing leaf and root area of inoculated plants. Growth promoting activity was also observed in field tests. The present study validates the potential use of A. oligosporus as a biocontrol of Meloidogyne spp. in tomato production systems under greenhouse. It also presents useful information on the use of different cultivation media and substrates for massive A. oligosporus spore concentrates.
Keywords: Arthrobotrys oligosporus, Meloidogyne, biocontrol, root system.
Phytoparasitic nematodes are considered one of the greatest threats to agriculture worldwide, with estimated losses exceeding $100 billion per year 1. Nematodes of the genus Meloidogyne are the essential phytoparasitic pathogens from an economic and scientific standpoint 2,3 as they can affect more than 2,000 species of plants 4, including cultivars of enormous food importance 5,6. Previous studies have described 100 species belonging to the genus Meloidogyne 7, of which M. incognita, M. javanica, M. hapla and M. arenaria have been reported in Ecuador 8,9, causing damage on more than 25% of the horticultural yield production of the country 10.
The management of Meloidogyne spp. is cumbersome as different reproductive strategies allow this species to adapt quickly to a wide range of temperatures and environments 11. This group's high diversity and the intensive crop production practices exacerbate their incidence and facilitate their dispersal across fields 12,13. These nematodes attack exposed roots by modifying their structure and growth, causing the formation of giant cells due to nutrient uptake 14, which translates, sometimes in a short time, into low yield 15.
With the growing advance of agriculture, chemical pesticides were developed 16 to overcome this problem. However, their frequent use has caused several side effects, affecting soil health by reducing biological activity 17. Another concern about pesticides is the risk they pose to human health, including infections, malignancies, lung inflammation, and others 18,19. New regulations regarding the use of these chemicals continue to be enforced by international agencies to help alleviate the negative effect of their use in farmers' lives 20. In this regard, the search for alternatives to mitigate the effect of nematodes less environmentally damaging and safer for human use is highly encouraged. Among those, biological control agents are a feasible option for pest management 21. Examples clearly have shown that controlling agents can reduce root-knot nematode populations and damage levels 22 up to 45% compared to uncontrolled conditions 23.
Within the gamma of antagonistic microorganisms, nematophagous fungi outstand this activity on trapping and devouring nematodes using specialized structures that allow them to penetrate the cuticle's nematode to grow inside, utterly killing them 24,25. Although their habitat is the soil, these fungi can change their lifestyle from saprophytic to pathogenic once they contact the nematode 26,27. Some reported modes of action include the development of specialized traps such as constriction rings, non-constriction rings, adhesive knobs, adhesive nets, and adhesive columns 28,29. Some more specialized species, such as Arthrobotrys oligosporus, can even produce volatile compounds to attracts their prey until the mycelial traps are ready to function correctly, which in some cases, can take more than twelve hours to form 30.
A. oligosporus grows in soil, around roots, and animal feces 31, but found particularly in nematode-infested soils 32. This fungus has significantly reduced pathogen populations 33. It has been extensively studied under laboratory and semi-controlled and open field conditions in tomato production 34,35. Thus, the search for new strains of A. oligosporus with similar or better nematophagous activity can help develop new products for controlling Meloidogyne spp. in Ecuador to improve tomato's local production. This study's objective was to evaluate the predatory activity of A. oligosporus strain C-2197 against the root-knot nematode Meloidogyne spp.
A. oligosporus strain C-2197 was isolated from decomposing leaves collected at the site of Santa Rosa, El Oro province (Ecuador), in a forest adjacent to a coffee plantation with limited anthropogenic activity. The samples were collected in plastic bags and taken to the laboratory, where they were placed in humid chambers for fifteen days 36,37. After the isolation and identification of this fungus using taxonomic keys, monoconidial colonies were obtained for later use. A. oligosporus C-2197 was deposited in the Microorganism Culture Collection of the Biotechnology Research Centre of Ecuador (CIBE) with the code CCMCIBE-H481. To maintain the predatory activity of our strain, the colonies were kept in cornmeal agar media (CMA) 38.
For this study, the initial population of Meloidogyne spp. was obtained from tomato plants of the Granja Agricola Experimental (GEA) located in the Escuela Superior Politécnica del Litoral (ESPOL). To maintain the nematode population, hybrid ABM-135 tomato plants were infected and kept in the greenhouse.
The growth of A. oligosporus C-2197 was evaluated in six different culture media: water agar, v-8 juice agar, corn meal agar (CMA), carrot agar, cornmeal + carrot agar, and oat agar. Radial growth was measured every 24 hours up to eight days. During this time, the Petri dishes were incubated at 28oC. Then, spore counts were performed in solutions obtained after scraping the colonies' surfaces with 5 ml of sterile water. The number of spores was calculated using a Neubauer camera. The experiment included five replicates per treatment.
To evaluate the predatory effect of A. oligosporus C-2197 under laboratory conditions, ten egg masses of Meloidogyne spp. were placed in Petri dishes with water agar. After 24 hours from the juvenile nematodes' appearance, 1 ml of spore solution of A. oligosporus C-2197, previously adjusted to 1x106 cfu/ml, was poured into each Petri dish. This trial included the evaluation of ten replicas (Petri dishes) placed at ambient temperature. After 24 hours of incubation, the number of live, dead, static, parasitized, and non-parasitized nematodes were evaluated.
To assess the effect of A. oligosporus C-2197 under greenhouse conditions, three-week old ABM-135 hybrid tomato plants were inoculated with ten egg masses of Meloidogyne spp. four days before placing the spore suspensions (1x106 cfu/ml) of the biocontrol. For this experiment, spore suspensions were obtained from colonies growing on a natural fiber substrate. The experiment was conducted following a completely randomized block design with four plants per treatment with four replicates (N=48), including T1 - A. oligosporus (AOL); T2 - Chemical control (NQ), and T3 - Water (CONTROL). Nematode root infection was evaluated by counting the number of egg masses and determining root-knot damage 39. Response variables included plant height, stem diameter, fresh and dry weight of the plant, fresh and dry weight of the roots.
Finally, the effect of A. oligosporus C-2197 was also evaluated in small field plots. Tomato plants previously infected with ten egg masses of Meloidogyne spp. and then planted in the field were treated with 1 L of a spore solution (1x106 cfu/ml) of A. oligosporus C-2197 sprayed directly on a "Bio-cover" (made of banana fiber woven with straw from rice ) (Figure. 1 A-B). The experiment included plants treated with a commercial product and negative control for proper comparison. In total, 10 plants were evaluated per treatment for six weeks following a completely randomized block design. Response variables included plant height, number of flowers and fruits.
Figure 1. (A) Inoculum production of A. oligosporus C-2197 in flasks under controlled conditions. (B) "Bio-cover" inoculated with A. oligosporus C-2197 surrounding hybrid tomato plants ABM 135 after 2 weeks of transplant in field plots.
The radial growth experiment showed that A. oligosporus C-2197 has a differential growth response in all media evaluated (Figure. 2A). The fungus thoroughly colonized the Petri dishes' surface, in some cases, from the fifth day. Of the six media evaluated, the best results were observed with oatmeal and cornmeal agar + carrot agar, however, no significant differences were found (Figure 2B). Sporulation of A. oligosporus C-2197 was highly increased with significant differences (p < 0.05) in v8 juice agar and corn meal agar CMA. Spore count in these treatments was higher than 1x106 cfu/ml after nine days of mycelium growth. In contrast, the fungus produced fewer spores on water agar and carrot agar (Figure 2C).
Figure 2. (A) Comparison of the growth of A. oligosporus C-2197 in different media, evaluated on the second, fourth and sixth day. (B) Radial growth of A. oligosporus C-2197 cultivated in six different media cultures: water agar, v-8 juice agar, corn meal agar CMA, carrot agar, cornmeal + carrot agar, and oatmeal agar. (C) Spore production of A. oligosporus C-2197 on different culture media.
The in vitro confrontation test revealed that A. oligosporus C-2197 had a control efficiency of 72.31% over the total number of nematodes exposed to the fungus. This percentage included dead and static nematodes (Figure. 3 A-B).
Figure 3. (A) Response percentage of the application of A. oligosporus against the nematode Meloidogyne spp. under in vitro conditions. (B) Nematodes trapped in three-dimensional webs of A. oligosporus C-2197
Control activity against Meloidogyne spp. was also observed in the greenhouse assay. Plants treated with A. oligosporus showed better root development than controls (Figure. 4) and significant differences in nematode egg mass reduction, with an effectiveness of 79%, compared to the negative control (Table 1). The plants' root health treated with A. oligosporus also correlated with the increase of plant height at the end of the experiment, which differed significantly from the controls (Figure. 5). Levels of root nematode infestation also differed among treatments. Plants treated with A. oligosporus and the commercial product showed less root-knot damage (values between 0-2) than the control (values 3-4), according to the qualitative scale used.
Field experiments also showed plants treated with A. oligosporus had better development (20 cm above control plants) (Table 2); however, there were no significant differences regarding the number of flowers and fruits produced by plants on each treatment.
Table 1. Effect of A. oligosporus on tomato plants inoculated with the root-knot nematode Meloidogyne spp. in greenhouse conditions.
Table 2. Evaluation of A. oligosporus C-2197 on some physiological and agronomic traits of tomato (Solanum lycopersicum) plants hybrid ABM 135
Figure 4. Greenhouse tests results. Evaluation of the root system.
Figure 5. Foliar development of tomato plants inoculated with Meloidogyne spp. and treated with A. oligosporus (center), commercial product (right), and control (left).
DISCUSSION AND CONCLUSIONS
In the present study, laboratory, greenhouse, and field tests were conducted to assess the activity of A. oligosporus C-2197 as a biocontrol for Meloidogyne spp. The in vitro tests showed that A. oligosporus C-2197 grew differently depending on the type of medium used, similar to previous reports 40,41. Our results are also consistent with previous work on the predatory activity of A. oligosporus controlling second instar juveniles of Meloidogyne spp. This fungus specializes in the formation of capture nets and demonstrates the ability to attract nematodes 42–45 and the production of toxins that paralyze the movement of juveniles 46.
Greenhouse tests confirmed the control activity of A. oligosporus C-2197 on the nematode population studied. This strain also showed growth-promoting activity for root development and leaf area. These data coincide with previous findings also reporting growing stimulus of plants after been treated with A. oligosporus, increasing production on tomato, rice, and carrot crops 23,34,47–49. Laboratory results showed that A. oligosporus C-2197 could develop well in substrates with high carbon and nitrogen ratios related to its saprophytic lifestyle, a necessary feature to establish in the soil outperform other saprophytic competitors 50,51. This characteristic facilitates the search for cheap substrates that can be implemented for massive scale production of a commercial product based on this strain 52.
In the present study, the application of A. oligosporus to the soil did not significantly affect nematode populations. More extensive field trials with A. oligosporus C-2197 are needed, as the "Bio-cover" may have affected the control of the fungus' efficacy. Evidence shows the efficacy of A. oligosporus to trap nematodes can vary when conditions are too variable 53–55.
In conclusion, it was demonstrated that A. oligosporus C-2197 showed significant efficacy in controlling nematodes in the laboratory and greenhouse, capable of reducing the target nematode population. Besides, A. oligosporus C-2197 had growth-promoting activity on tomato plants. This study shows the potential use of A. oligosporus C-2197 for Meloidogyne spp's biocontrol, specifically as a management alternative for tomato production in greenhouse conditions.
The authors are grateful to Dr. María Isabel Jiménez for the technical guidance to isolate nematodes from plants. The project supported this research "Isolation, identification and characterization of beneficial microorganisms associated with cocoa cultivation and their potential as biological control agents" from Milagro State University - UNEMI.
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Received: 2 december 2020
Accpted: 25 january 2021
Adela Quevedo1, Marcos Vera-Morales1, Fernando Espinoza-Lozano1, Rafael F. Castañeda-Ruiz3, Daynet Sosa1,2, Freddy Magdama1,2*
1 ESPOL Polytechnic University, Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador, Campus Gustavo Galindo Km. 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador.
2 ESPOL Polytechnic University, Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ciencias de la Vida, Campus Gustavo Galindo Km. 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador.
3 Instituto de Investigaciones Fundamentales en Agricultura (INIFAT), Tropical ‘Alejandro de Humboldt’, OSDE, Grupo Agrícola, Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200
*Corresponding author: email@example.com