Evaluation of Stropharia sp. 1.2052 nematicidal effects against Meloidogyne incognita on tomato

To use unique nematicidal mechanism of Stropharia, the strain 1.2052 was evaluated for its potential to control the root-knot nematode Meloidogyne incognita on tomato. In vitro, the inhibition rate in 24 h of Stropharia sp. 1.2052 isolate was 100% for second-stage juveniles of M. incognita and Panagrellus redivivus, 41.81% for Caenorhabiditis elegans and 99.25% for Bursaphelenchus xylophilus, respectively. In pot experiments, isolate 1.2052 reduced the root-knots to 68.16 to 84.19% in one month and 45.28 to 88.24% in two months after treatment, respectively. The reduction of nematodes in soil ranged from 26.39 to 61.18% compared to the negative control. There were significant efficacies for control of root knot nematodes on tomato.


INTRODUCTION
Root-knot nematodes (Meloidogyne spp.) cause serious damages to agriculture and forest. It has been reported that plant parasitic nematodes cause annual losses of several billion of crops worldwide (Koenning et al., 1999;Perry et al., 2009). Various fungi have been tested and used to control root-knot nematodes during the last decades. The most studied fungi used and applied to control nematodes are Hirsutella rhossiliensis and Pochonia chlamydosporia. H. rhossiliensis is one of few endoparasitic fungi of mobile stage of nematode. Both species produce infective, adhesive conidia that may be attached to the cuticle of a passing nematode, and has shown great potential in soybean cyst nematode control (Li et al., 2008). P. chlamydosporia has a worldwide distribution, and has been found in nematode suppressive soils to parasitize nematode eggs (Manzanilla-Lo´pez et al., 2013). Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii were studied for their effects on pathogenicity of M. incognita race 2 in soybean (Akinsanmi et al., 2003). Kiewnick et al. (2006) reported that a Paecilomyces lilacinus strain was selected to control M. incognita. Arthrobotrys dactyloides was tested against M. incognita (J2) on Tomato (Kumar et al., 2006). Muscodor albus was evaluated as a potential biocontrol agent against four plant-parasitic nematodes of economically important vegetable crops (Riga et al., 2008). Biological control of root-knot nematode (M. javanica) by Trichoderma harzianum BI was also investigated in greenhouse and laboratory experiments (Sahebani and Hadavi, 2008).
Species of Stropharia (Strophariaceae, Basidiomycota) have purple-brown to tobacco brown spores and appear in woods, grasslands, compost piles, and animal dung. Stropharia cultures can produce unique stellate cells, called acanthocytes. Farr (1980) has made a detailed study of the morphology and development of acanthocytes, and considered the ability to form acanthocytes as a characteristic of the whole genus (Norvell et al., 2000). Luo et al. (2006)  and Bursaphelenchus xylophilus within hours on agar plates.
The nematode-attacking activity of this fungus is carried out by spiny acanthocytes and their mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, suggesting that acanthocytes are functional in soil. In the present study, mechanism of Stropharia sp. 1.2052 activities against a population of the root-knot nematode M. incognita was evaluated on tomato, in greenhouse experiments.

Fungal culture
Stropharia sp. 1.2052 was collected from forest in Yunnan, P. R. China. The pure culture was isolated from fruiting body, identified by Luo et al. (2006), and deposited in the culture collection of Key Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, P. R. China.
Solid culture of this strain was prepared on wheat grains. Wheat was soaked 24 h with tap water and 1 h with boiling water, later mixed with CaCO3, glucose and sawdust (wheat, 60%; sawdust, 36%; CaCO3, 2%; glucose, 2% v%). Such culture medium was transferred in 250 ml flask and sterilized at 121°C for 2 h. Inoculated cultures were incubated at 25°C for 30 days when more than 80% wheat was consumed.

M. incognita
the nematodes population was collected from tobacco in Yunnan, P. R. China. Tobacco roots with egg masses were dipped in water to remove adhering soil, cut into 0.5 to 1 cm pieces and agitated in 0.5% sodium hypochlorite solution for 2 to 3 min. Egg masses were rinsed with sterile water then placed in 0.5% sodium hypochlorite for 2 to 3 min, agitated and rinsed with sterile water (Kerry and Bourne, 2002). The eggs were incubated at 25 ± 1°C for three to five days to obtain second stage juveniles (J2) with modified Bearmann funnel method (Gray, 1984). The J2 were then used for in vitro and pot experiments.

Panagrellus redivivus
Nematodes were cultured on oatmeal medium (oatmeal: 20 g, water 80 ml) at 25°C for seven days, and then refrigerated at 4°C prior to use.

Bursaphelenchus xylophilus
Botrytis cinerea was cultured on a potato dextrose agar (PDA) plate at 25°C until the fungus was fully grown. The plate was then inoculated with the pine nematode, and then cultured until the fungal mycelia had been completely consumed.

Caenorhabiditis elegans
The nematodes were mutiplied in oatmeal medium at room tempe-rature (21 to 26°C) for 6 to 7days.

In vitro experiment
Washed nematodes (80 to 100) were transferred to the PDA culture plates containing abundant well-developed hyphae of Stropharia sp. 1.2052. The plates were incubated at 25°C, counting the numbers of active and inactive nematodes under a stereomicroscope (50×) at different times (24, 48, 72, 96, 120 and 144 h). The nematodes were considered dead if they showed no response to physical stimuli. Uninoculated plates were used as negative control, and avermectin (0.8 μg/ml) was used as positive control. Each treatment was replicated three times, and the mortalities (%) were calculated.

Pot assay
One-month-old tomato seedlings were transplanted into 15 cm-diameter pots containing 1000 g sterilized soil (loam soil : humus soil : sand, 1:1:1,v/v ). A week after transplanting, the soil around the seedlings was inoculated with M. incognita at 2000 juveniles per plant. Pots were arranged in a randomized block in a greenhouse at 25 ± 2°C.
Four treatments were applied: three concentrations of strain 1.2052 (50, 75 and 100 g) were incorporated with soil around the seedlings. Avermectin (0.5%, 0.02 g/pot) was used as positive control, and untreated pots were used as negative control. The experiment was completely randomized with three replicates. All pots were kept under greenhouse conditions (25 to 32°C) and watered when needed.
After 30 and 60 days, the plants were harvested. Nematode damage was measured by rating root galls on the 0 to 5 scale (0 = complete and healthy root system, no infection, 1 = very few small galls only be detected upon close examination, 2 = 25% of root system severely galled, 3 = 25 to 50% of root system severely galled and not, 4 = 50 to 75% of root system severely galled, 5 = >75% of root system severely galled, nearly no healthy roots). In addition, population of nematodes in soil, plant heights, and shoot fresh weights were determined.

Statistical analysis
Results were tested with SPSS version 17.0 by one-way analysis of variance (ANOVA). Significantly differences between treatment means were separated by using t test at the P<0.05 (Sahebani and Hadavi, 2008).

Effect of fungi on nematode in vitro
Application of Stropharia sp.1.2052 significantly inhibited nematodes motility in vitro, when compared to control in water. At 24 h complete inhibition was observed for all the J2 of M. incognita and for P. redivivus, whereas lower values were found for B. xylophilus and C. elegans, which required a longer exposure to reach the same level of inhibition (Table 1).

Effect of fungi on M. incognita in pot assay
Significant control of root-knot nematodes was observed Means of three replicates ± SD. Values in each column followed by * one and ** two asterisks were significantly different from water control at P≤0.05 and P≤0.01, respectively. Means of three replicates ± SD. Values in each column followed by * one and ** two asterisks were significantly different from water control at P≤0.05 and P≤0.01, respectvely.
in pots. A dose response relationship was observed for treatments with Stropharia isolate 1.2052 which caused the greatest decrease of galls in one and two months, compared to positive controls. Application of Stropharia at lower doses resulted in lower damage reduction in one and two months, respectively (Table 2). There were significant reductions of the M. incognita J2 (Table 2), however a dose-response relationship was less evident as the treatment with 75 g/pot caused the highest reduction of J2 from roots, either in one and two months, compared to negative control. Application of Stropharia sp. 1.2052 at 50 and 100 g had 8 and 5/g roots in one month, 25 and 21/g roots in two months, respectively. The percent reduction of nematodes in soil compared to the negative control appeared dose-dependent, and was higher than avermectin.
Differences among treatment were observed for plant height and biomass. The plants showed an increase in height after two months, with tallest plants recorded for treatment with Stropharia sp. 1.2052 at 75 g/pot and the shortest from the positive control. The fungus application significantly improved shoot weight over the inoculated treatment, the highest weight recorded for treatment at 75 g/pot. A dose-response relationship was also observed for Stropharia sp. 1.2052 applied at 50, 75 and 100 g/pot that increased the fresh root weights, compared to positive and negative controls, respectively (Table 3).

DISCUSSION
Many mushrooms, previously considered only as saprobes, are actually considered capable of taking advantage of other organisms present in their microcosm, exploited for nutritional purposes (including nematodes, plants, other fungi, and bacteria). There is a special group of nematophagous basidiomycetous fungi provided with hyphal appendages that are indispensable for their nematode-attacking ability. The appendages within the various species include hourglass-shaped adhesive knobs on members of the genus Nematoctonus (Drechsler, 1949(Drechsler, , 1954, and its teleomorph Hohenbuehelia (Barron and Dierkes, 1977). Other appendages include the secretory cells of Pleurotus spp. (Barron and Thorn, 1987), the secretory appendages of Conocybelactea (Hutchison et al., 1996), the stephanocysts on Hyphoderma spp. (Burdsall, 1969;Tzean and Liou, 1993) and the spiny balls produced by Coprinus comatus (Luo et al., 2004). Thapa et al. (1987) reported that Stropharia rugoso-annulata was resistant to the mycophagous nematode species Aphelenchoides sacchari and Ditylenchus myceliophagus. However,  Grewal (1990) found that the mycophagous nematode Aphelenchoides composticola was able to feed on S. rugoso-annulata. Stropharia species also have been reported to be able to immobilize the nematode P. redivivus and B. xylophilus with spiny acanthocytes and the results shown that the acanthocytes are functional in soil (Luo et al., 2006). In the present study, a series of experiments were undertaken to further investigate the effect of Stropharia sp. 1.2052 against nematodes. In the in-vitro assay, this isolate inhibited more than 80% of motile stages of M. incognita, P. redivivus and B. xylophilus, except C. elegans. Application of Stropharia sp. 1.2052 to infested tomato plants in the greenhouse reduced the number of galls and final nematode population of M. incognita with highest efficacy either at one and two months. Such results are similar to the nematode control effects reported for many other fungi. In controlled experiments on tomato, a pre-planting soil treatment with P. lilacinus strain 251 was reported to reduce root galling by 66% (Kiewnick et al., 2006). When F. oxysporum was inoculated on soybean, the mean number of nematodes that penetrated roots decreased by 75%, when the soil was treated with S. rolfsii the number decreased by 78% (Akinsanmi et al., 2003). Greenhouse study shows that M. albus caused significant reduction (91 and 100%) of Paratrichodorus allius in soil, and 100% for Pratylenchus penetransin bean roots and soil; 85 and 95% for Meloidogyne chitwoodi in potato roots; 100% for Meloidogyne hapla both in pepper roots and soil (Riga et al., 2008). The trapping fungus A. dactyloides was applied with cow dung manure, which reduced the number of root knots by 61.7 to 66.6% and of juveniles by 68.1 to 88.0% (Kumar et al., 2006).

Conclusion
Stropharia sp. 1.2052 provided effective control of nematode comparable to those reported for other fungal species. As Stropharia spp. are common saprophytic basidiomycete in soil, they have less chances to be pathogenic to plants and human beings, compared to other fungi. The main problems in application of Stropharia are related to its cultivation and form of delivering into soil. Its use as symbiont of seeds or plant roots could also be considered for future application, as well as its development in the integrated management of root-knot nematode.