In vitro evaluation of nematophagous activity of fungal ...

Environment Health Techniques Nemathophagous activity in vitro

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Research Paper In vitro evaluation of nematophagous activity of fungal isolates Lidia Nicola, Solveig Tosi and Dario Savini Mycology Laboratory, Department of Earth Science and Environment, University of Pavia, Pavia, Italy

Four filamentous fungi associated with nematodes were isolated and identified from litter samples collected in the Integral Natural Reserve “Bosco Siro Negri” (PV, Italy): Arthrobotrys dactyloides, Arthrobotrys oligospora var. oligospora, Pochonia bulbillosa, and Pochonia chlamydosporia var. catenulata. Their capacity to break down the nematode population was evaluated in vitro by means of simple and reproducible multiwell plates method. All fungal strains were able to cause a death-rate significantly different from the controls (p < 0.05). Precisely, A. dactyloides caused, on average, a 26% death rate increase in the nematode population compared to the control, A. oligospora var. oligospora 25%, P. bulbillosa 12%, and P. chlamidosporia var. catenulata 17%. The method has also allowed to determine the more active fungi as regards the prey’s life cycle stage. The most active strains against nematodes (adults) were A. dactyloides and A. oligospora var. oligospora, known to attack adults or larval stages by means of tridimensional traps. On the contrary P. bulbillosa and P. chlamydosporia, known to attack mainly the nematode life stage of cysts, showed lower activity against adult nematodes. Keywords: Nematode-attacking fungi / Arthrobotrys / Pochonia / Biological control Received: July 27, 2012; accepted: October 26, 2012 DOI 10.1002/jobm.201200431

Introduction There is an urgent need for new technologies of crop protection. Low toxicity to humans and wildlife, low environmental impact, low residues in food, and compatibility with integrated pest management (IM) programs are increasingly important factors in the selection procedure of new pesticides. Nowadays modern society values sustainable farming systems because of their potential in protecting wildlife and biodiversity and in reducing environmental harm caused by intensive farming practice [1]. Biological control is suggested as a promising alternative method to chemicals, although a large-scale use is still far to be achieved. The term “biological control” was proposed by Tubeuf in 1914 and by Smith in 1919, concerning plant pathogens and insects respectively. Some authors want to include also genes and Gabriel and Cook [2] reported a broad definition of “biocontrol” as “the use of organisms, genes, or gene Correspondence: Dr. Solveig Tosi, Mycology Laboratory, Department of Earth Science and Environment, University of Pavia, via S. Epifanio 14, 27100 Pavia, Italy E-mail: [email protected] Phone: þ39 0382984870 Fax: þ39 038234240 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

products to control undesirable organisms (pests) and favor desirable organisms such as crops, trees, and beneficial microorganisms and insects”. The interest in “biological control” has increased during the years and the number of papers on this topic increased exponentially from about 15 in 1960 to 200 in 1980 [3]. Parasitic nematodes are one of the most dangerous pests mainly for their capacity of producing very resistant cysts. Nematophagous fungi have been considered as potential biological control agents for plant parasitic nematodes, since Zopf [4] observed that the nematode-trapping fungus Arthrobotrys oligospora Fresen. was able to capture and colonize motile nematodes. A. oligospora and A. superba Corda were the basic constituents of a commercial product against nematode species of Meloidogyne [5]. A. oligospora together with Pochonia chlamydosporia (Goddard) Zare & W. Gams are the biocontrol agents of the commercial product Pochart (ELEP, Cornaredo, Milano, Italy). Chlamydospores of P. chlamydosporia (strain PcMR) are the constituent of the bionematocide PcMR-1, used against the plant parasitic nematodes. In this paper the capacity of attacking nematodes was evaluated in vitro and compared in four filamentous fungi isolated from leaf litter of an integral natural reserve in Italy.

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J. Basic Microbiol. 2013, 00, 1–5

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Materials and methods Samples’ typology and origin The samples, which the fungi were isolated from, were represented by leaf litter collected in the Integral Natural Reserve “Siro Negri,” near Zerbolò (PV, Italy). This woodland, on the Ticino river, belongs to the University of Pavia and the access is restricted to researchers. At the beginning of June 2010, 10 samples were aseptically collected, kept in sterile plastic bags at 5°C and processed within 3 days. Fungal strain isolation, maintenance, and identification The nematophagous fungi were isolated according to the sprinkled plate technique [6] with some modifications. Leaf litter samples (1–2 g) were deposited onto the center of a water-agar or corn meal agar plate. Plates were incubated at room temperature (25°C) to the daily light and controlled after 5 days for three weeks. Fungi were isolated with a sterile needle when they were observed in association with nematodes (in the so-called “nematodes’ graveyard”). Among the isolated fungal strains, four of the most common were identified on the basis of their morphodimensional characteristics using slide microcultures. Nematodes collecting and counting Nematodes, easy to be found in soil, belonging to the saprotrophic populations, were used to evaluate the fungal isolates’ nematophagous activity. Leaf litter samples were sprinkled on water-agar plates and incubated at 25 °C for 2 or 3 days. After this period a great increase in the nematodes population was observed. The plate was gently “washed” with sterile distilled water (2–3 ml). The suspension obtained was centrifuged at 750 RPM for 2 min and the precipitated nematodes harvested. A population of nematodes belonging to the genus Mononchus was easily bred and maintained at 25°C on water agar feeding on soil-borne bacteria naturally present in the suspension. Nematodes were collected and washed three times in sterile distilled water before performing the tests. From this suspension, 5 µl were placed on a slide and the number of nematodes was counted under the microscope (10). A volume proportionally containing 100 nematodes was collected and adjusted to 1 ml. Test for the evaluation of the nematophagous activity In vitro test was a modification of Bedelu’s et al. [7] microwell plate assay. The tests were carried out in 24microwells plates. Spore suspensions were prepared from 10–15 days fungal culture plates, washing the colony’s ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

surface with sterile distilled water and adjusted at 0.5 McF of turbidity by means of a nephelometer (Vitek Systems ATB 1550, bioMerieux Marcy l’Etoile, France). In each well, a volume of 600 µl of the conidial suspension for each fungus was distributed together with a volume of 1 ml of sterile water containing 100 nematodes. The control was prepared with 1 ml of nematodes suspension and 600 µl of sterile distilled water. Each test was performed in five replicates. The microwell plates were incubated at 25°C. After 72 h slides were prepared using 8 µl of the microwell contents and observed under the stereomicroscope (magnification 5). The nematodes mortality was checked, counting the living nematodes (such as the moving nematodes, easier to be observed); nematodes’ degradation and presence of fungal colonization were evaluated. Statistical analysis On the basis of the surviving nematodes for each experimental replication (R ¼ 5) a data matrix was set up for the data statistic analysis. All the statistic analyses were carried out with the computer package Minitab Student Edition 12.0 [8]. Analysis of Variance test (One way ANOVA), that compares the differences between the averages of three or more independent groups, was used. Afterwards the Tukey test was applied to detect any significant difference between single treatments (average number of survived nematodes after the contact with the fungi) and the control (microwells without nematophagous fungi).

Results Fungal strains isolation and identification Ten nematode-associated fungal strains were isolated from the leaf litter samples from the Siro Negri Natural Reserve. The isolates belong to the imperfect fungi group, that produce conidiophores and conidia and a hyaline or pink mycelium. The following species were identified by morphodimentional analysis: Pochonia bulbillosa (W. Gams & Malla) Zare & W. Gams, P. chlamydosporia var. catenulata (Kamyschko ex G.L. Barron & Onions) Zare & W. Gams, Arthrobotrys dactyloides Drechsler and A. oligospora var. oligospora Fresen. Evaluation of the nematophagous fungi’s activity The modified Bedelu’s et al. [7] microwell plate assay used to evaluate the nematophagous fungal activity, gave the results reported in Fig. 1. The test’s results showed an average of dead nematodes from 70% to 84% in the treatments with the fungal

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Table 1. One-way ANOVA for the comparison among the average values of surviving nematodes ml1 in the treatments and in the controls. Source

DF

SS

MS

F

p-Value

Treatment Error Total

4 20 24

904.64 102.4 1007.04

226.16 5.12

44.17

0

DF, degrees of freedom; SS, sum of the squares; MS, mean square; F, value of the test; p-value, significance of the test.

Figure 1. Average of dead nematodes (dark gray) or surviving nematodes (light gray) after 72 h of treatment with the four nematophagous fungal strains in comparison to the control.

strains; in the control the mortality was 58%; this high value will be discussed later. The percentages of dead nematodes due to the activity of the fungi can be calculated as the differences between the treatments and the control. The net values ranged from 12% to 26%. The boxplot in Fig. 2 shows the average, and the relative variability of the datum (number of living nematodes ml1) after the treatments with the fungal strains. All the four treatments differed from the control (26 2 ind. ml1), with lower average values of living nematodes (min: 10 3 ind. ml1 with A. dactyloides, max: 19 3 ind. ml1 with P. bulbillosa). After verifying the necessary normality (Anderson Darling normality test: A-Sq.: 0.45; p > 0.05) and equality of variances (Batlett test: Bt ¼ 0.10; p > 0.05), we could statistically verify the differences among the nematodes on treatment and control nematodes, applying the parametric test: Analysis of Variance (One-Way, ANOVA; Table 1). Each treatment was significantly different from the control as supported by the Tukey test analysis (Table 2).

Figure 2. Average number of surviving nematodes after treatment with nematophagous fungal strains (in light gray) compared to the control (in dark gray). ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Moreover the nematode populations treated with A. oligospora and A. dactyloides showed a higher mortality in comparison with those treated with P. chlamydosporia and P. bulbillosa as confirmed by Tukey test. In order to have a confirmation on the four strains’ nematophagous activity, some slides were observed under the optical microscope. In the treatments with A. dactyloides and A. oligospora var. oligospora several nematodes were observed associated with traps. In the treatments with P. chlamydosporia var. catenulata and P. bulbillosa nematodes were deteriorated, or colonized by fungal hyphae.

Discussion In this work an in vitro test that allows the screening of fungal strains as potential biocontrol agents against nematodes and the measure of their virulence was evaluated. The test is based on the use of microwell plates and it reminds of the method used to calculate the minimal inhibitory concentration (MIC), commonly used to evaluate the fungicide activity of chemicals and extracts. The strains’ activity was evaluated basing on number of living nematodes after 72 h with the fungus. The most active fungal strains against nematodes were A. oligospora var. oligospora and A. dactyloides, causing an average net death rate of 25% and 26%, respectively, if compared to mortality recorded in the control. The quite high (58%) mortality of the control may be due to the maintenance of the nematodes for 72 h in water only before slides observation. Nonetheless, the death rates in the microwells inoculated with the fungal isolates were significantly higher than in the control. The data obtained in the microwells treated with A. oligospora var. oligospora and A. dactyloides are consistent with the observation of many nematodes trapped in the mycelium. The activity of A. oligospora var. oligospora is in accordance with the literature concerning data recorded in field experiments [9–11]; the results obtained on A.

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Table 2. Tukey test, multiple comparison. The values in the cells are the test confidence intervals.

A. oligospora A. dactyloides P. bulbillosa Control

P. chlamydosporia

A. oligospora

A. dactyloides

P. bulbillosa

0.720 9.28 1.32 9.88 7.480 1.08 15.08 6.52

3.680 4.88 12.48 3.92 20.08 11.52

13.08 4.52 20.68 12.12

11.88 3.32

The differences between the treatments and the control are not significant (p > 0.05) when the intervals contain the zero ().

dactyloides are similar to those on A. oligospora var. oligospora but they are not in accordance with the literature that, on the contrary, shows a limited efficiency of this fungus in field experiments [12, 13]. The activity of the two Pochonia strains was higher than the control, but lower than the one observed for the Arthrobotrys strains. The results were confirmed by the microscope observations: nematodes were colonized by both fungi. P. chlamydosporia var. catenulata caused a death rate increase of 17% compared to the control; the activity observed is in accordance with the literature that reports a strong activity of this species in field experiments [14, 15]. This fungus is known to attack especially the nematodes at the stage of egg, as demonstrated by Braga et al. [16] on P. chlamydosporia against Oxiurus equi, a horse parasite. P. bulbillosa caused an average nematodes’ net mortality of the 12% compared to the control’s death rate. Association with nematodes and activity data concerning this species are scarce and are reported only for nematode eggs [17, 18]. Nematophagous activity of P. bulbillosa reported in this paper represents a new datum, giving new insights on the biology of the fungus. The Arthrobotrys strains tested were more active than the Pochonia ones, at least in the experimental condition where the preys considered were nematode adults or larvae stages; this observation can be explained by the fact that P. chlamydosporia and P. bulbillosa are particularly active against the eggs as reported by the literature [16, 18]. This underlines also the good sensitivity of the assay used, that was able to indicate the best nematophagous strain for killing nematodes (adults and larvae stages). The experiments underlined also the good virulence of the Pochonia strains against adult nematodes, although it wasn’t as high as the Arthrobotrys strains. Further researches could be carried out by means of this method, to evaluate the efficacy of associated nematode fungi not yet deeply investigated such as P. bulbillosa. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Acknowledgments We wish to thank the Italian Ministero dell’Ambiente e della Tutela del Territorio e del Mare for the financial support. We also wish to thank the unknown Reviewer, who improved the manuscript with very useful suggestions.

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