Phanerochaete chrysosporium strain B-22, a nematophagous fungus parasitizing Meloidogyne incognita

The root-knot nematode Meloidogyne incognita has a wide host range and it is one of the most economically important crop parasites worldwide. Biological control has been a good approach for reducing M. incognita infection, for which many nematophagous fungi are reportedly applicable. However, the controlling effects of Phanerochaete chrysosporium strain B-22 are still unclear. In the present study we characterized the parasitism of this strain on M. incognita eggs, second-stage juveniles (J2), and adult females. The highest corrected mortality was 71.9% at 3 × 108 colony forming units (CFU) mL-1 and the estimated median lethal concentration of the fungus was 0.96 × 108 CFU mL-1. Two days after treatment with Phanerochaete chrysosporium strain B-22 eggshells were dissolved. A strong lethal effect was noted against J2, as the fungal spores developed in their body walls, germinated, and the resulting hyphae crossed the juvenile cuticle to dissolve it, thereby causing shrinkage and deformation of the juvenile body wall. The spores and hyphae also attacked adult females, causing the shrinkage and dissolution of their bodies and leakage of contents after five days. Greenhouse experiments revealed that different concentrations of the fungal spores effectively controlled M. incognita. In the roots, the highest inhibition rate for adult females, juveniles, egg mass, and gall index was 84.61%, 78.91%, 84.25%, and 79.48%, respectively. The highest juvenile inhibition rate was 89.18% in the soil. Phanerochaete chrysosporium strain B-22 also improved tomato plant growth, therefore being safe for tomato plants while effectively parasitizing M. incognita. This strain is thus a promising biocontrol agent against M. incognita.


Introduction
Infections due to plant-parasitic nematodes lead to global agricultural economic losses of more than $157 billion [1] via reducing the quality and quantity of greens. Root-knot nematodes (Meloidogyne spp.), which comprise 98 species and parasitize almost every species of vascular plants [2], are the most economically important and destructive obligate plant-parasitic nematodes. They occur globally, especially in tropical and subtropical agricultural areas, and cause a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and it commonly inhabits forest litter and fallen trees. This fungus has strong degradation potential for organic compounds owing to its diverse extracellular oxidative enzymes in the growing hyphal mass [24], and it can efficiently depolymerize, degrade, and mineralize all components of plant cell walls, including cellulose, hemicellulose, and the more recalcitrant lignin [25]. Moreover, the inoculation of P. chrysosporium in greenhouse soils has been greatly effective for reducing root-knot nematode diseases and the relative disease index of root-knot nematodes on cucumber [26]. The use of P. chrysosporium in the soil can also control root wilt disease by Fusarium oxysporum and improve plant physiological status [27]. Pleurotus ostreatus, another white-rot fungi, can produce toxin droplets to attack and digest nematodes [28,29]. The gene sequences encoding fruiting body lectins of Pleurotus cornucopiae are similar to that encoding the lectin of a nematode-trapping fungus [30].
The present study aimed to evaluate the efficacy of P. chrysosporium strain B-22 on the biocontrol of M. incognita under in vitro and field conditions, as this was confirmed as the main plant-parasitic nematode at the greenhouses in Taigu (Shanxi Province, China), and to assess the safety of this strain for plant growth. Our results provide basis for the development of P. chrysosporium strain B-22 as a biopesticide for the control of M. incognita in greenhousegrown vegetables. Further, the results reported herein will allow the development of new strategies for the theoretical and practical management of M. incognita.

Nematode inoculum preparation
Infected tomato roots were collected from a tomato plant grown in a greenhouse in the city of Taigu (Shanxi Province, China), and a single egg mass was cultured on tomato as inoculum to establish the nematode population for the experiment. The species of nematode was identified as M. incognita based on morphological and morphometrical characters [31]. Egg masses were directly extracted from infected root galls using 1% sodium hypochlorite (NaClO), and the separated eggs were gently washed with sterile water to remove the NaClO [32]. Egg masses were kept in distilled water in the dark at 25˚C for 48 h. The hatched juveniles were counted under a stereomicroscope (Nikon Instruments Inc., Tokyo, Japan). The suspensions of M. incognita were diluted to approximately 200 juveniles per milliliter with distilled water. After surface sterilization, eggs, juveniles, and females were stored at 4˚C for subsequent trials.

Phanerochaete chrysosporium inoculum preparation
P. chrysosporium B-22 was obtained from the Plant Pathology Department at Shanxi Agriculture University in China and was cultured on potato-dextrose agar medium. P. chrysosporium was morphologically identified as in Burdsall [33]. Five days after incubation at 25˚C, purified P. chrysosporium B-22 were used to produce spore suspensions for inoculation; these spore suspensions were further adjusted with autoclaved distilled water to obtain 3 × 10 8 colony forming units (CFU) mL -1 for treatments, which were counted using a hemocytometer [34].
the density adjusted to 100 females mL -1 . Egg masses were added into sterile Petri dishes with sterile water and incubated at 25˚C ± 1˚C for 3 days in the dark to hatch M. incognita J2. These were then surface sterilized with 1% NaCOl and incubated to obtain a 1,000 J2 mL -1 suspension. Egg masses were centrifuged in 1% NaOCl at 2,000 rpm for 3 min, and free eggs were collected to prepare a 3,000 eggs mL -1 suspension.
The parasitic effects of strain B-22 on the eggs of M. incognita were determined as described by Zhang et al. [35]. Briefly, 100 μL of M. incognita egg suspension (about 300 eggs) was placed in a sterile Petri dish containing strain B-22 spore suspension at 3 × 10 8 CFU mL -1 . A blank control was prepared with an equal volume of sterile water instead of the strain B-22 spore suspension. Petri dishes were incubated at 25˚C ± 1˚C for three days. Samples were taken for microscopic examination using a Nikon 80i microscope with an image analysis system (Nikon Instruments Inc.).
The parasitic effects of strain B-22 on M. incognita J2 were determined according to Schwartz [36]. Briefly, 100 μL of M. incognita J2 suspension (about 100 J2 individuals) was placed in a sterile Petri dish containing strain B-22 spore suspension at 3 × 10 8 CFU mL -1 ; these Petri dishes were incubated at 25˚C ± 1˚C for 1 day, after which M. incognita J2 were removed and placed on a glass slide with 4% agarose for examination by fluorescence microscopy with an image analysis system (Nikon 80i microscope; Nikon Instruments Inc.). Microscopic examinations were performed 2 days later.
The parasitic effects of strain B-22 on M. incognita adult females were determined according to Dong et al. [37]. Briefly, strain B-22 was grown on water agar (WA) until the colony diameter reached near the edge of the dish. Sterile coverslips were placed in the WA, and 100 μL of adult female suspension (about 10 females) was transferred onto the coverslips and incubated at 25˚C ± 1˚C for five days. A control group was prepared without strain B-22. Samples were subjected to microscopic examination using the Nikon 80i microscope with an image analysis system (Nikon Instruments Inc.).
The mortality of M. incognita J2 was determined by visual inspection of stiff individuals that stayed motionless after gently poking with a bamboo needle. The results were used to calculate the mortality and corrected mortality rates (%) of M. incognita J2. The median lethal dose (LC 50 ) of strain B-22 conidia for M. incognita J2 was determined by simple linear regression analysis of the conidial concentration of strain B-22 and corrected mortality rate among J2 of M. incognita: y = ax + b, where "y" is the corrected mortality rate among J2 of M. incognita and "x" is the conidial concentration of strain B-22. Mortality (%) = (number of dead J2 in each treatment/total number of tested J2) × 100%. Corrected mortality (%) = (mortality in treatment-mortality in control)/ (1-mortality in control) � 100%.

Greenhouse evaluation of the control efficacy of P. chrysosporium strain B-22
Phanerochaete chrysosporium strain B-22 was incubated in potato dextrose agar plates at 25˚C ± 1˚C for 10 days, after which 5 mL of sterile water was pipetted onto the surface of the plates, and the spores were scraped off from the plates [38]. The spore suspension was then filtered through a fine-mesh screen (diameter 0.15 mm) to separate the spores from the hyphae, and stored at 4˚C until use.
A greenhouse experiment was laid in a completely randomized design with six replications (pots) using autoclaved soil sterilized at 121˚C for 2 h. The soil was individually transferred into the plastic pots (4 kg soil pot -1 ), and three seeds of the M. incognita-susceptible F1-hybrid tomato cultivar JG9002 were planted in each pot. When tomato seedlings were growing, each pot was treated with the above spore suspension at the following concentrations: 1.875 × 10 7 , 3.75 × 10 7 , 7.5 × 10 7 , 1.5 × 10 8 , and 3 × 10 8 CFU mL -1 . After 15 days, every pot was artificially inoculated with 1,000 M. incognita J2. All pots were maintained in the greenhouse at 25˚C, under 16 h sunlight and at 65% relative humidity. Pots were fertilized weekly with approximately 3 g L -1 of Poly Fertisol (N:P:K = 14:10:14) and watered daily as needed. Pots inoculated with M. incognita J2-but not with the P. chrysosporium strain B-22 spore suspension-served as controls, while pots that were not inoculated with M. incognita J2 nor the P. chrysosporium strain B-22 spore suspension served as blanks.
After 10 weeks, plant and soil samples from all treatments were randomly collected and brought to the laboratory for counting nematodes in the roots and soil, and calculating rootknot index. The count of nematodes in the soil was determined as in Castillo et al. [39], and root count was determined as in Sharon et al. [40]. The extent of root galling damage was determined as previously described [41,42]. The severity of tomato root galling was assessed on a scale from 0 to 10, where 0 = no knots on roots and 10 = all roots severely knotted or no more root system. Root-knot index = S (number of diseased plants in each rating � score/total number of plants investigated � highest rating) � 100%. Six replications of each treatment were included.

Safety of P. chrysosporium strain B-22 on tomato plant growth
Plant height, root length, aboveground fresh mass, and root fresh mass were measured independently in each treatment and control after 10 weeks of growth. Plant height and root length were measured with a graduated ruler, while plant fresh mass was weighed on an electronic balance.

Statistical analyses
All experiments were repeated six times. Data were processed using Microsoft Excel 2007 (Microsoft Corp., Redmond, WA, USA) and expressed as means ± standard deviation (n = 6). The significance of differences in the counts of females, egg masses, and juveniles in the roots, tomato root gall index, and measurements of plant height, root length, aboveground fresh mass, and root fresh mass were examined using the t-test and one-way analysis of variance in SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was considered at p < 0.05.

Parasitic effect of P. chrysosporium strain B-22 on M. incognita
Phanerochaete chrysosporium strain B-22 parasitized the eggs of M. incognita after only two days of treatment. In the initial period of parasitism, the spores of P. chrysosporium strain B-22 were in contact and conglutinated with the eggs, and then geminated and produced short hyphae around them (Fig 1A). The fungus grew rapidly, causing the aggregation of the inner contents of the eggs, and its hyphae ran through the eggs causing shrinkage of the egg shell. More hyphae grew from the egg (Fig 1B). Over the following two days, the eggshell was deformed and shrunk further. This continued until the eggshell was completely dissolved by the fungus. The eggs were broken by the hyphae. At last, the dense mycelia of the fungus enveloped the eggs, which at the time looked abnormal and misshapen (Fig 1C). In the controls that were not inoculated with the conidia of strain B-22, the eggs of M. incognita were intact; microscopic examination confirmed that they had a smooth surface and uniform contents (Fig 1D).
Phanerochaete chrysosporium strain B-22 also parasitized M. incognita J2. After three days of incubation, P. chrysosporium strain B-22 spores were seen on the cuticle of M. incognita J2 upon microscopic examination, as observed for eggs. The conidia of the fungus developed in the body wall of nematode juveniles (Fig 2A), and the geminated spores produced hyphae from the body of the nematodes. Thus, P. chrysosporium strain B-22 grew on the surface of the cuticle of M. incognita J2 (Fig 2B). With time, P. chrysosporium strain B-22 produced more mycelium on M. incognita J2 and dissolved their cuticles, causing shrinkage and deformation of their body wall (Fig 2C). After five days of incubation, microscopic examination revealed that the cuticles of M. incognita J2 were dissolved or severely deformed. Dissolved residues of the bodies of M. incognita J2 were also clearly visible, and the cuticle was bent and shrunken ( Fig 2D). Eventually, the fungus produced massive spores that developed a dense mycelium and parasitized the body surface of the nematode juveniles. The color of the cuticles was extensively altered (Fig 2E). In uninoculated controls, M. incognita J2 showed intact body wall and slow movement (Fig 2F).
Phanerochaete chrysosporium strain B-22 also parasitized M. incognita adult females. Upon four days of incubation, the spores of P. chrysosporium strain B-22 surrounded M. incognita adult females and stuck to their surfaces. The contents of the bodies of the females leaked out, and short hyphae grew out from their bodies (Fig 3A). After seven days of treatment, P. chrysosporium strain B-22 formed dense hyphae crossing the body walls of M. incognita adult females. Moreover, the adult female body looked severely atrophied and it was partly dissolved due to leakage (Fig 3B). In uninoculated controls, M. incognita adult females showed complete and healthy bodies with a smooth surface and obvious boundaries. Body contents of the adult females were also intact ( Fig 3C).

Lethal effect of P. chrysosporium strain B-22 on M. incognita J2
Phanerochaete chrysosporium strain B-22 conidial concentration and treatment time significantly influenced the mortality of M. incognita J2 (Table 1). Compared with uninoculated M. incognita J2, the mortality of M. incognita J2 infected with the fungus suspension was significantly higher (p < 0.05) in all treatments, from 1.875 × 10 7 to 3 × 10 8 CFU mL -1 . In addition, mortality increased linearly with conidial concentration and time after treatment. Twentyfour hours of treatment were sufficient for causing M. incognita J2 mortality. Corrected mortality rate reached 42.8% under treatment with 3 × 10 8 CFU mL -1 conidial concentration and it was significantly different (p < 0.05) from the other treatments and from the control after 24 h. Further, corrected mortality was up to 54.5% after 48 h. The highest corrected mortality (71.9%) was observed after 72 h under treatment with a conidial concentration of 3 × 10 8 CFU mL -1 . This mortality rate was significantly different (p < 0.05) from all other treatments.

Greenhouse evaluation of P. chrysosporium strain B-22 control efficacy against M. incognita
The conidial concentration of the P. chrysosporium strain B-22 suspension significantly influenced the different stages of M. incognita ( Table 2). The antagonistic effect of the different Phanerochaete chrysosporium B-22 parasitizes root-knot nematodes conidial concentrations on the nematode was significantly greater (p < 0.05) for all treatments relative to blanks and controls. Overall, P. chrysosporium strain B-22 significantly decreased (p < 0.05) the numbers of M. incognita eggs, J2, and adult females, as well as the root gall index. The number of M. incognita J2 in the soil also decreased due to treatment with the fungal suspension. The best control efficiency on M. incognita was obtained at the conidial concentration of 3 × 10 8 CFU mL -1 , which was significantly different (p < 0.05) from all other treatments. The inhibition ratio of M. incognita adult females ranged between 46.15% and 84.61% in the roots, while that of the juveniles ranged from 45.57% to 78.91%, and that of the eggs from 50.39% to 84.25%. Furthermore, the inhibition ratio of the root gall index was 33.33%-79.48%; in the soil, the inhibition ratio of M. incognita J2 reached 68.51%-89.18% (Table 2).

Safety of P. chrysosporium strain B-22 for tomato plant growth
Soil inoculation with P. chrysosporium strain B-22 was found to be safe for plant growth; in fact, this fungus significantly promoted tomato plant growth (Table 3). Plant height, root length, aboveground fresh mass, and root fresh mass were all significantly higher (p < 0.05) upon treatment with P. chrysosporium strain B-22 at different conidial concentrations, compared with blanks and controls. The highest promotion effect was achieved at the conidial concentration of 3 × 10 8 CFU mL -1 , which was significantly different (p < 0.05) from all other treatments. In this case, plant height was 29.5 cm, root length was 30 cm, aboveground fresh mass was 9 g, and root fresh mass was 1.5 g, while the corresponding values in the controls were 9.7 cm, 9.1 cm, 1.5 g, and 0.28 g, respectively. The highest increase rates for plant height, root length, aboveground fresh mass, and root fresh mass were 202.06%, 185.71%, 426.66%, and 292.85%, respectively (Table 3).

Discussion
Phanerochaete chrysosporium is a white-rot fungus that has been identified as nematophagous [43]. We isolated P. chrysosporium strain B-22 as a nematophagous fungus lethal to M. incognita. Previous studies on P. chrysosporium have focused on removing toxic environmental pollutants [44]. In addition, its inoculation greatly reduced the disease index of root-knot nematode in the field and prevented wilt disease in cucumber [26] and for the control of Fusarium wilt of cut chrysanthemum [27]. Because P. chrysosporium produces many extracellular enzymes [45], it has been used in the biodegradation of lignin and nicotine in tobacco stalk [46], as well as polycyclic aromatic hydrocarbons [45] and other pollutants [47,48]. The present study identified P. chrysosporium as a parasitic fungus of M. incognita, which was When M. incognita eggs were treated with P. chrysosporium strain B-22, the fungus parasitized the eggs by producing hyphae, which first surrounded the eggshell and then destroyed the eggs. Some studies have shown that the egg-parasitic fungi Verticillium suchlasporium, P. lilacinus, Pochonia spp., and T. harzianum can secrete protease and chitinase, which degrade certain cyst nematode proteins to effectively destroy the nematode eggshell and later parasitize and kill the eggs [49]. As protein and chitin are the main chemical constituents of nematode cuticles and eggshells, the infection of M. incognita eggs by P. chrysosporium strain B-22 might also be involved in eggshell decomposition through the production of protease and chitinase.
P. chrysosporium strain B-22 also parasitized M. incognita J2 by producing sticky conidia that enveloped the juveniles. These were active initially but became completely destroyed after five days of incubation. Adhesive conidia of P. chrysosporium strain B-22 first stuck to the nematode body wall and produced germ tubes to invade it. Then, massive hyphae grew and crossed the nematode cuticles, causing the deformation and death of the juveniles by leakage. Moreover, the external body wall was gradually disintegrated, further indicating that P. chrysosporium strain B-22 might indeed be able to produce proteases and chitinase to degrade the nematode cuticle, as reported for the parasitic fungi Lecanicillium psalliotae [50]. Nematophagous P. ostreatus, another white-rot fungus, produced toxin droplets to attack nematode in 2-4 h, and its hyphae disintegrated the nematode body after 24-48 h [28,43,51,52]. It is unclear whether P. chrysosporium strain B-22 employed a similar mechanism to attack M. incognita J2 juveniles prior to invasion and degradation. The decomposed relics of the external body wall of M. incognita J2 well after death indicated that this fungal strain might produce some substance(s) that kill M. incognita J2 juveniles.
When parasitizing M. incognita adult females, P. chrysosporium strain B-22 produced mycelial masses on their body surfaces. The parasitizing mycelia then crossed and digested the adult females. Our results indicated that P. chrysosporium strain B-22 parasitized M. incognita and thus effectively worked as a biocontrol agent. The mechanism of P. chrysosporium strain B-22 for controlling M. incognita involved the production of sticky conidia and hyphae to parasitize M. incognita, and on secreted extracellular enzymes and possibly other substances to digest the body of the nematode.
Overall, the present study evidenced that P. chrysosporium strain B-22 was nematophagous toward M. incognita. However, it is not clear whether other root-knot nematode species can be digested by this fungus, as the host range of nematophagous species is specific. Pleurotus ostreatus can trap and consume Aphelenchus avenae but can then be consumed by Filenchus misellus. Tylencholaimus parvus was susceptible to both P. pulmonarius and P. ostreatus whereas A. avenae was resistant to both P. pulmonarius and P. ostreatus. Oscheius tipulae was susceptible to P. pulmonarius but resistant to P. ostreatus. Metarhabditis rainai was resistant to P. pulmonarius but susceptible to P. ostreatus [53]. Thus, the nematophagous effects we found for P. chrysosporium strain B-22 may be limited to M. incognita.
Results from our greenhouse experiments clearly demonstrated that different concentrations of P. chrysosporium strain B-22 could control M. incognita in tomato. The control efficacy increased with conidial concentration of the fungal suspension. Moreover, treatment with P. chrysosporium strain B-22 significantly increased plant height, root length, and aboveground mass and root fresh mass of tomato plants. Thus, this strain effectively controlled M. incognita while improving the growth of the host tomato plants. These results indicate that P. chrysosporium strain B-22 is safe for greenhouse-grown tomato seedlings.

Conclusions
This study characterized the parasitism of P. chrysosporium strain B-22 on the eggs, J2, and adult females of the root-knot nematode M. incognita. We also evaluated the control efficacy and safety of this fungal strain when applied to tomato plants cultured in a greenhouse. The results demonstrated the potential of P. chrysosporium strain B-22 as a promising biocontrol agent for M. incognita. However, further studies are needed to characterize the compounds responsible for the toxic effect of this parasitic fungal strain on the nematode, its physiological characteristics, and its control efficacy against other root-knot nematodes.