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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 1 | Page : 68-74
Mushrooms as efficient biocontrol agents against the root-knot nematode, meloidogyne incognita
Gaziea Soliman1, Waill Elkhateeb2, Ting-Chi Wen3, Ghoson Daba2
1 Department of Plant Pathology, Nematology Unit, Agriculture and Biology Research Division
2 Department of Chemistry of Natural and Microbial Products, Pharmaceutical Industries Researches Institute, National Research Centre, Giza, Egypt
3 State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering; The Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education; The Mushroom Research Centre, Guizhou University, Guiyang, China
Correspondence Address:
Waill Elkhateeb
Department of Chemistry of Natural and Microbial Products, Pharmaceutical Industries Researches Division, National Research Centre, El Buhouth Street, Dokki, Giza 12311
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/epj.epj_80_21
Background Root-knot nematodes in general and Meloidogyne incognita, in particular, are pests that cause agricultural losses. Currently, nematode control relies on chemical nematicides, which are hazardous to the environment and human health. The increasing demand for ecofriendly nematicides has prompted researchers to look into biocontrol agents that act as efficient and long-lasting alternatives to the currently used chemicals.
Objective The aim of the study was to evaluate the in-vitro nematicidal activity of eight mushroom (Cordyceps militaris, Metacordyceps neogunnii, Hericium erinaceus, Dictyophora indusiata, Cerioporus squamosus, Tirmania nivea, Tirmania pinoyi, and Agaricus impudicus) extracts against M. incognita juveniles and eggs.
Materials and methods Hydromethanolic extracts were prepared from the fruiting bodies of mushrooms under investigation. Then the obtained extracts were evaluated for their in-vitro nematicidal activity against M. incognita juveniles’ second stage after 24, 48, and 72 h of treatment, also against their eggs.
Results and conclusion All tested mushroom extracts were capable of inducing mortality in M. incognita second stage juveniles with mortality percentages ranging from 79.3 to 97%. On the other hand, the tested mushroom extracts exhibited some nematostatic and nematicidal activity against M. incognita eggs as compared with the control after 7 days using 80 µl/ml concentration. The tested mushroom extracts caused suppression in M. incognita eggs hatching with inhibition rates that ranged from 59.38 to 81.25%. A. impudicus hydromethanolic extract showed the highest inhibition as compared with the control and other tested mushroom extracts as it caused a relative suppression that reached 81.25% against M. incognita eggs after 7 days of exposure. The same mushroom extract has achieved a juveniles mortality of 97%. A. impudicus extract is nominated as a potential nematicidal agent. Further studies are required to confirm the potency of this extract and analyze its chemical profile.
Keywords: biocontrol, egg hatching, juveniles, Meloidogyne incognita, mushroom extracts, nematicidal
Root-knot nematodes, Meloidogyne spp., are serious pests that cause wide-ranging losses tto different crops worldwide, and are ranked among the most difficult agricultural pests to control due to their wide host range, short life cycles, and high reproduction rate [1],[2]. Meloidogyne incognita is a highly polyphagous nematode that affects crops in warmer temperatures and can be found in greenhouses in both hot and temperate countries [3]. In past decades, the use of chemical nematicides was one of the primary means of controlling root-knot nematodes [4]. However, chemical nematicide strategies are losing favor among researchers worldwide due to their high cost, effect on human health, resulting pollution, and serious environmental impacts, which led to searching for alternative approaches for sustainable crop production, control plant diseases, and plant-parasitic nematodes. Biological control is a sustainable alternative to chemical control including using natural and environment-friendly components to reduce plant-parasitic nematodes [5],[6],[7],[8],[9],[10]. Mushrooms are generous sources of compounds that can act as anticancer, antioxidant hypocholesterolemic, and wound-healing agents [11],[12],[13]. Moreover, many mushrooms have a vital role in the biological control of plant-parasitic nematodes as alternatives to chemical nematicides due to their nematicidal properties, as it is easy to obtain and cultivate, as well as being ecofriendly and sometimes edible [14],[15]. The bioactive compounds produced by the mushrooms have a selective effect, preserving nontarget species, implying the possibility of developing environmentally friendly goods [16],[17]. Cordyceps militaris (orange caterpillar mushroom) and Metacordyceps neogunnii are entomogenous medicinal mushrooms [12],[18],[19]. However, few studies are available reporting the biological activities of M. neogunnii extract. On the contrary, C. militaris is a well-studied mushroom that can be cultivated artificially and hence its bioactive compounds can be obtained in good quantities [20]. Hericium erinaceus is another well-known medicinal mushroom that exerts various biological activities such as antimicrobial, antiproliferative, antitumor, hemagglutinating, hypoglycemic, antiaging, and immunomodulatory effects [12]. Dictyophora indusiata is also a well-studied medicinal mushroom that is known centuries ago and has potential use as a functional food [21],[22]. Both Tirmania nivea and Tirmania pinoyi are edible desert truffles (ectomycorrhizal wild mushrooms) that grow under certain environmental conditions and exist in different surrounding locations including the north coast of Egypt, Saudi Arabia, and Yemen. They are ethnically edible for their medical and nutritional values [23],[24]. On the other hand, Cerioporus squamosus (dryad’s saddle or pheasant’s back mushroom) is a medicinal mushroom that has been studied and its extract showed antimicrobial, antioxidant, anticancer, and wound-healing properties [25]. The last mushroom investigated in this study is the basidiomycetes mushroom Agaricus impudicus (tufted wood mushroom). Studies published on this mushroom mostly discuss its habitat, taxonomy, and morphology [26]. However, publications describing the bioactivity of its extract are relatively rare.
The study aims to evaluate the in vitro nematicidal activity of the hydromethanolic extracts of the mushrooms C. militaris, M. neogunnii, H. erinaceus, D. indusiata, C. squamosus, T. nivea, T. pinoyi, and A. impudicus as biocontrol agents against M. incognita mortality and egg hatchability.
Materials and methodsMushroom sample extraction
Mushrooms tested in this study (C. militaris, M. neogunnii, H. erinaceus, D. indusiata, C. squamosus, T. nivea, T. pinoyi, and A. impudicus) ([Figure 1]) were provided by Prof. Dr Waill Elkhateeb, Prof. Dr Ting-Chi Wen, and Prof. Dr Abdu ALKolaibe. Identification of samples was conducted by Prof. Dr Waill Elkhateeb and Prof. Dr Ting-Chi Wen. Hydromethanolic extracts of mushrooms were individually prepared as described by Elkhateeb et al. [25]. Briefly, 250 g of fruiting bodies of each mushroom was cut into small pieces (1 cm). Then, the pieces were kept for 2 days in Erlenmeyer flasks that contain 80% methanol at room temperature. Samples were filtered and the resulting filtrates were concentrated at 37°C using a rotary evaporator (Heidolph, Germany). The obtained extracts were stored at 4°C in a clean closed container until further use.
Figure 1 Fruiting bodies used to prepare hydromethanolic extracts of mushrooms: Agaricus impudicus (a), Cordyceps militaris (b), Dictyophora indusiata (c), Tirmania nivea (d), Tirmania pinoyi (e), Metacordyceps neogunnii (f), Hericium erinaceus (g), and Cerioporus squamosus (h). Photos were taken by Prof. Dr Waill Elkhateeb, Prof. Dr Ting-Chi Wen, and Dr Ghoson Daba.In-vitro nematicidal activity of hydromethanolic extracts of mushrooms
Inoculum of Meloidogyne incognita
The pure population of M. incognita was obtained from greenhouse culture on tomato plants maintained at the Plant Pathology Department, National Research Centre, Egypt. After 40 days, the plants were uprooted, and the roots were washed free of soil and cut into 2 cm pieces. The nematode eggs were extracted from infected roots of tomato in 0.5% sodium hypochlorite on a 25-µm sieve according the method of Hussey and Barker [27]. The extracted eggs were used immediately for egg hatching ability test, or incubated for egg hatching for 72 h at the laboratory temperature (25±3°C) using the modified Baermann technique [28] to provide second-stage nematode juveniles (J2) for juvenile’s mortality test. To estimate the inoculum density, the egg suspension was poured into a measuring cylinder. The numbering of eggs was estimated in five aliquots of 1 ml in a Hawksley counting slide under a light microscope and their five means were calculated. For concentrating the egg suspension, it was left to settle down for several hours, afterward the extra water was decanted off leaving the bottom undisturbed and the same was done with J2 to estimate the inoculum density. The percentage J2 mortality and inhibition of egg hatch of the root-knot nematode M. incognita were used to test the in-vitro nematicidal activity.
Nematicidal activity of mushroom hydromethanolic extracts on Meloidogyne incognita J2 mortality
The nematicidal activity of hydromethanolic extracts of the eight mushrooms against M. incognita juveniles was tested using two concentrations (40 and 80 µl/ml). One milliliter from each concentration had 100±5 M. incognita J2 in it, separately. They were arranged in a completely randomized design at 25±3°C after 24 and 72 h of exposure. All treatments were conducted in five replicates and the average results were compared with distilled water as control. Also, after 72 h of exposure, the juveniles were washed in distilled water and transferred to aerated distilled water for 24 h. The juveniles that did not regain their activities and those do not move when probed with a fine needle were considered ‘dead.’ On the other hand, the juveniles were considered active when they were visibly flexible and then the average percentages of nematode recoveries were determined [29]. The number of active and inactive J2 was counted with the aid of a microscope and the percentages of inactive larvae were calculated to evaluate the percentage of juveniles’ mortality.
The percentages of nematode mortality were calculated according to Abbott’s formula [30] as follows:
Juvenile mortality (%)=(m–n)/(100–n)×100, where m and n indicate the percentages of mortality in treatments and control, respectively.
Effect of mushroom extracts on the inhibition of Meloidogyne incognita egg hatch
In this experiment, the direct effect of the eight mushroom hydromethanolic extracts on M. incognita egg suppression was investigated at one concentration (80 µl/ml). One milliliter contain about 100±5 M. incognita J2 with an 80 µl/ml final concentration. Plain water in the test tube supplied with nematode suspension was served as control. Treatments were conducted in five biological replicates compared with the negative control. The test tubes were kept at room temperature 25±3°C. Hatching eggs were examined, after 7 days of exposure; the number of hatched eggs was counted under a stereomicroscope by using a special microscopic slide for counting nematodes. After 1 week, the total number of J2 was calculated. The relative suppression rate was calculated as follows: relative suppression rate (%)=(number of J2 in control−number of J2 in treatment)/number of J2 in control×100.
Data analysis
Data obtained from laboratory bioassays for hatched eggs and mortality (%) were subjected to analysis of variance and means compared according to Duncan’s multiple range test [31].
ResultsNematicidal potential of the mushroom species
The nematicidal activity of eight mushroom extract species against M. incognita juveniles were tested at two concentrations (40 and 80 µl/ml) in in vitro was tests. The obtained results, after 24 h of exposure, revealed that all mushroom extracts caused significant mortality in M. incognita J2 compared with control. The percentage of mortality ranged between 67 and 78.00% at 80 µl/ml concentration in all mushroom extracts, while the untreated control achieved zero. T. nivea achieved the highest percentage of mortality, 73.00 and 78.00%, at two concentrations, 40 and 80 µl/ml, respectively ([Figure 2]). After 48 h, the results showed that the tested concentrations were extremely hazardous to nematode juveniles. T. pinoyi extract had the highest mortality of 88.67% when tested at 80 µl/ml. After 72 h of exposure, all mushroom extracts cause significant mortality in M. incognita J2 as compared with control. The percentage of mortality ranged between 79.3 and 97% on using 80 µl/ml concentration in all mushroom extracts tested. C. militaris had the highest percentage of mortality, 92.0 and 97%, at two concentrations of 40 and 80 µl/ml, respectively ([Figure 3] and [Figure 4]). The percentage of nematode mortality differed with concentrations of mushroom extracts and exposure time. The percentage mortality of second-stage juveniles (J2) exposed to highly concentrated mushroom extracts was markedly greater than that of those exposed to low-concentration mushroom extracts and controls after 24, 48, and 72 h of incubation. Microscopic investigation after treatment with extracts of C. militaris, M. neogunnii, H. erinaceus, D. indusiata, C. squamosus, T. nivea, T. pinoyi, and A. impudicus revealed no changes in the nematode bodies of juveniles treated with mushroom extracts (data not shown).
Figure 2 Effect of hydromethanolic extracts of mushrooms on juvenile mortality of Meloidogyne incognita after 24 h. Error bars represent mean±SD.
Figure 3 Effect of hydromethanolic extracts of mushrooms on juvenile mortality of Meloidogyne incognita after 48 h. Error bars represent mean±SD.
Figure 4 Effect of hydromethanolic extracts of mushrooms on juvenile mortality of Meloidogyne incognita after 72 h. Error bars represent mean±SD.Inhibitory activity of mushroom extracts on Meloidogyne incognita eggs
In the in-vitro experiments, all the tested mushroom hydromethanolic extracts had some nematostatic and nematicidal activity against M. incognita eggs as compared with the control. Generally, different mushroom extracts remarkably suppressed the hatching of M. incognita eggs. However, inhibition rates ranged from 59.38 to 81.25% after 1 week of exposure. Data presented in [Table 1] revealed that A. impudicus caused the greatest inhibitory effect (81.25%) followed by C. squamosus (79.17%), H. erinaceus (78.12%), C. militaris (66.67), T. nivea (64.58%), M. neogunnii (63.52%), and C. militaris (59.38%), after 7 days of exposure as compared with control ([Table 1] and in [Figure 5]).
Table 1 Inhibition activity of mushroom extracts against Meloidogyne incognita eggs after 7 days of exposure
Figure 5 Effect of hydromethanolic extracts of mushrooms on suppression hatchability of Meloidogyne incognita eggs. Discussion M. incognita is one of the most destructive and widely distributed plant-parasitic nematodes attacking roots of different economic crops such as sugarcane, okra, potatoes, tobacco, tomato, and cotton [32]. As much as 40% yield losses in Indian tomato was caused by M. incognita. In 2015, M. incognita caused losses in American cotton yield that reached 1.35% of total cotton production [33]. The use of chemicals for controlling pests, and their impact on human health and the environment had forced scientists to search for ecofriendly alternatives to face the economic loss caused by pests without polluting the environment and compromising the whole ecosystem. Hence, screening for biocontrol solutions is of serious importance to fight such harmful pests.
In this study, we have evaluated the antinemic effect of some mushrooms’ hydromethanolic extracts [34]. Microscopic investigation after treatment with extracts of C. militaris, M. neogunnii, H. erinaceus, D. indusiata, C. squamosus, T. nivea, T. pinoyi, and A. impudicus revealed no changes in the nematode bodies of juveniles treated with mushroom extracts. This indicates that mushroom extracts could have the ability to paralyze and kill plant nematode juveniles. This action could be related to the production of paralyzing compounds existing in these extracts. Similar findings were observed [34], where the mushroom Pleurotus cystidiosus secretes paralyzing toxins that affect the mobility of nematodes. Generally, mushrooms secrete various secondary metabolites which have nematotoxic activity [35],[36],[37],[38]. For example, the total number of biopesticides isolated from basidiomycetes is considerably high [39]. Many Pleurotus-originated fatty acids were also reported for their nematicidal action [17]. This can be causing the nematicidal effect recorded for C. militaris, M. neogunnii, H. erinaceus, and D. indusiata where fatty acids represented the majority of compounds in their chemical profiles [12],[19]. On the other hand, the mushroom Pleurotus ostreatus was reported to produce a nematode toxin similar to peroxides that killed 95% of free-living adult worms of Panagrellus redivivus and phytopathogenic nematode Bursaphelenchus xylophilus [40]. Also, many antioxidants are produced by wild mushrooms, including phenolic acids, flavonoids, and ascorbic acid [41]. It should be noted that there is a positive relationship between phenolic compounds and nematode mortality [42],[43],[44]. The hatching inhibition potential and nematicidal effect of mushroom extracts on M. incognita eggs were confirmed in in-vitro experiments. Mushroom extracts and cell-free culture broth contain some bioactive compounds such as polysaccharides, proteins, fats, minerals, glycosides, alkaloids, volatile oils, phenolics, and flavonoids are reported to possess antinemic effects against plant nematodes. Accordingly, the antinemic impact of mushroom extracts or filtrate on eggs of M. incognita could be attributed to the intrinsic nature of toxic metabolites such as phenols and fatty acids.
In light of the current difficulties in managing plant-parasitic nematodes, extracts of mushrooms could be a potential management strategy for controlling plant nematodes. Sangeetha et al. [45] found that the bioactive compounds of Ophiocordyceps sinensis inhibited egg hatching (94%) and juvenile mortality (92%) in M. incognita after 72 h of incubation. Similarly, the ethanolic extract of P. ostreatus exerted high nematicidal activity against Meloidogyne spp. that reached 99.1% after 1 h of treatment. On the other hand, aqueous extracts of the mushrooms Boletus sp., Lactarius deliciosus and Amanita muscaria caused mortality rates that ranged between 90.7 and 100.0% after 1 day of exposure [46]. In another in-vitro study, 10 mushroom aqueous extracts suppressed the hatching of second-stage nematode juveniles [47],[48]. In the laboratory, the root-knot nematode, M. incognita juveniles were reduced by using aqueous extract of the mushroom P. ostreatus [15]. The use of bioactive compounds from a luminous mushroom (Neonothopanus nambi) for the control of plant-parasitic root-knot nematode M. incognita was also described [16].
ConclusionsNovel ecofriendly biocontrol agents are urgently needed to replace the currently utilized toxic nematicides, if we are to avoid further damage to the earth’s ecosystems. Mushrooms are a potential alternative to these chemicals. They have also been found to protect against plant-parasitic nematodes. Results showed in the current study nominate extracts of A. impudicus, C. squamosus, and H. erinaceus as the most potent extracts in terms of increased percentage mortality of juveniles and reduced hatchability of eggs. After 72 h, juvenile mortality achieved 97, 95.67, and 95.67%, respectively, while, suppression rate of eggs for the same species extracts recorded 81.25, 79.17, and 78.12%, respectively, after 1 week. While these findings are encouraging, further field and mechanistic studies and chemical analyses are required to confirm the potency and mechanism of action of these extracts as nematicidal agents.
AcknowledgementsThe authors thank Prof. Dr Abdu Ghalib ALKolaibe, Microbiology Department, Faculty of Science, Taiz University, Yemen, for providing some of the mushroom samples.
Availability of data and materials: The data that support the findings of this study are available from the corresponding author, Waill Elkhateeb, on reasonable request.
Authors’ contributions: All the authors have read and approved the final manuscript. W.E. and G.D. have substantially contributed to the conception and design of the work. W.E.: collection, identification, and extraction of mushroom samples. T.C.W.: collection and identification of mushroom samples. G.S.: execution of nematodal experiments, analysis, and interpretation of results, writing and reviewing of the manuscript. G.D.: extraction of mushroom samples, writing, and reviewing of the manuscript.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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