Evaluation of Pochonia chlamydosporia ( Goddard ) Isolates for Suppression of Meloidogyne incognita , Root-Knot Nematode of Tomato

Restricted applications of chemical nematicides has directed to the development of ecofriendly alternatives. Culture filtrates in different concentrations (20, 40, 60, 80 and 100%) of the six isolates of Pochonia chlamydosporia were evaluated for their nematicidal activity. Significant variations were observed in the inhibitory activity of culture filtrates against egg hatching and second stage juveniles of M. incognita at different concentrations. The culture extract of PC-6 showed maximum inhibition of egg hatching followed by PC-1. On the other hand PC1 isolate caused high mortality of second stage juveniles followed by PC-6. Significant variation among the isolates was observed for parasitism of M. incognita juveniles and eggs. Among the tested isolates, PC-1 and PC-6 were selected for the further studies based on in vitro egg inhibition and larval mortality. Both PC1 and PC6 applied at the rate of 6 × 10 chlamydospores per gram of soil per plant reduced the population of nematode and improved the growth and productivity of plant under screen house and field conditions. Different growth parameters like plant height, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight were measured. Efficacy of isolates was different in winter and summer crop. No significant difference among the treatments was observed in summer crop. However in winter crop, considerable effect of both isolates was observed. Fungal application was effective at the rate of 6 × 10 chlamydospores per gram of soil per plant in both winter and summer crops. Root coating with chlamydospores suspension significantly reduced number of galls on roots. Little effect of both isolates was observed with mycelial root coating. No lesions were seen on P. chlamydosporia inoculated roots of tomato plants.


Introduction
Root-knot nematode (Meloidogyne incognita) is a major and endemic pathogen of tomato crop in northern part of Khyber Pakhtunkhwa province of Pakistan (Musarrat, Firoza, & Shahina, 2006).Average crop losses due to this pathogen in vegetable crops may reach up to 50-80% (Ornat & Sorribas, 2008;Sikora & Fernandez, 2005).Characteristic symptoms of infected plant are knots or galls on roots at the point of infection (Agrios, 2004).These galls disrupt the vascular tissue of roots that interfere with uptake of water and mineral and translocation of photosynthate resulting in stunted growth, chlorosis and wilting of the plant (Palomares-Rius, Escobar, Cabrera, Vovlas, & Castillo, 2017).Chemical control is a common mean of controlling plant diseases (Hirooka & Ishii, 2013;Duponnois, Chotte, & Sall, 2001).However, chemicals penetrate poorly into roots tissue to contact nematode eggs.They also can cause environmental problems (Garima, Singh, & Trivedi, 2005;Ploeg, 2002;Kerry, 2000).The use of resistant cultivars is a valuable component in Root-knot nematode management but in many important crops their resistant cultivars are not available.Moreover the effectiveness of some resistant cultivar is restricted to a few races of the nematode (Briar, Wichman, & Reddy, 2016).Meloidogyne incognita has a wide host range, which makes crop rotation impracticable.Thus, for controlling this nematode, long term integrative approches and development of alternatives of chemical pesticide is urgently needed to protect our crops from the risk of this pathogen (Martin, 2003).The nematophagous fungus P. chlamydosporia (Goddard) is an egg nematode parasite and has been found as an effective in biological management of cyst and Root-knot nematodes (Kerry & Bourne, 2002).The fungus survives in the soil as chlamydospores (Manzanılla-Lopez et al., 2013) and develops colonies around nematodes eggs and attached with eggs shells through appressoria (Lopez-Llorca, Macia-Vicente, & Jansson, 2008).Then the fungus penetrates into egg by physical pressure and enzymatic activities of different enzymes including proteases, lipases, chitinases and collagenases (Tikhonov, Lopez-Llorca, Salinas, & Jansson, 2002;Kopcke, Wolf, Anke, & Sterner, 2001).P. chlamydosporia isolates showed variation in their bio control efficiency depending on different factors i.e., fungal growth rate and its initial population in the rhizosphere, chlamydospores and enzymes productions, availability of water and fungal growth supported by the host plant species (Manzanilla-López et al., 2013;Bourne & Kerry, 2000).Utilization of biological agents in a management of Root-knot nematode in tomato crop is a good alternative of chemical pesticides in places infested with root-knot nematodes.Therefore the present study was designed for the collection of isolates of P.chlamydosporia from different region of Khyber Pakhtunkhawa, Pakistan and to evaluate their efficacy against M. incognita.

Survey and Sampling
Root-knot nematodes field were surveyed for isolation of P. chlamydosporia.About 150 roots and rhizosphere soil were randomly collected from 35 different locations of Khyber Pakhtunkhwa province of Pakistan.Samples were sealed in plastic bags and brought from field to laboratory at the department of Plant Pathology, the Agriculture University and stored in refrigerator at 4 °C until use.

Separation of Root-knot Nematodes From Roots
Eggs and females of root-knot nematode were separated from the gall roots, collected randomly from field following standard method of Barker et al. (1985).Briefly, pieces of gall root were rinsed with tap water for 3-5 min to remove the soil, cut into small pieces and then blended in an electric blender.Nematode eggs were collected on 26-μm-aperture sieve while the females on 75 μm sieve.The females and eggs were stored in 1% saline solution in separate beakers (Hussey & Barker, 1973).

Identification and Mass Production of P. chlamydosporia
Sodim hyphochloride solution (NaOCl 1%) was used for surface steralization of females and egg masses for 30 seconds to relaese eggs.After sterilization the eggs were rinsed three times with sterile distilled water.The eggs were then put on semiselective medium developed by Kerry et al. (1993).The composition of media per liter was 17 g corn meal agar (Oxoid), 37.5 mg carbendazim, 75 mg rose bengal, 37.5 mg thiabendazole, 17.5 mg NaCl and 3 ml Triton X 100, supplemented with 0.2 g/L of streptomycin sulfate and 5 mg/L of penicillin.The plates were incubated at 25 °C.After three days, P. chlamydosporia isolates were identified (Barnett & Hunter, 1998) and then grown on grains for mass production of P. chlamydosporia.Extraction of chlamydospores was done using standard method of (Crump & Kerry, 1981).

Culture Filtrate (CF) of Pochonia chlamydosporia
Small agar pieces (4-5 cm) of fresh culture of P. chlamydosporia were put in to 250 ml Erlenmeyer flasks containing sterile potato dextrose broth.The flasks were sealed and incubated at 25 °C for 10 days on a rotatory shaker.The filtrate was centrifuged at 8,000 rpm for 20 minutes.Mycelial mat, hyphae and spore were removed passing through sterile Whatman filter paper (0.2 µm) (Kerry, 1986) and stored at 4 °C.

Single Species Nematode Culture
Single egg mass and identified female of Meloidogyne incognita was collected and inoculated in 3 weeks old nursery seedlings of tomato cv.Money maker (tomato)were raised in earthen pots containing sterilized soil.The egg masses were collected 50 days after inoculation and re-cultured them.

Effect of Culture Filtrate on Egg Hatching and J 2 Mortality
Effect of culture filtrate of P. chlamydosporia isolates on M. incognita was determined by using standard method described by (Mukhtar & Pervaz, 2003).Different concentrations (20, 40, 60, 80 and 100%) of culture filtrate were pepared.Approximately 100 eggs of M. incognita from freshly cultured inoculum were treated with 1% sodium hypochlorite (NaOCl) solution for 30 seconds and then transferred to into 5 cm diameter petri plates containing 5 ml of each dilution of culture filtrate.Eggs in sterilized distilled water were used as control.The treatments were replicated six times at intervel of 24, 48 and 72 hours.For testing the effect of culture filtrate on J 2 mortality, five milli-litre (5 ml) of each dilution of the culture filtrate was poured in 5 cm diameter petri plates.
Each petridish was plated with 50 fresh hatched second stage juveniles of M. İncognita.Sterilized distilled water having juviniles was used as control.The treatments were replicated six times.Effect of culture filterate was checked at 24, 48 and 72 hrs.Under a stereomicroscope the the number of live or dead juveniles were counted by probing nematodes with a fine needle to differentiate paralyzed or dead nematodes from live ones (Ayatollahy, Fatemy, & Etebarian, 2008).The assay was repeated twice.Percentage of juvenile mortality was noted (Sun, Gao, Shi, Li, & Liu, 2006).

Parasitism of Eggs and J 2
Five isolates of P. chlamydosporia were tested against M. incognita eggs and J 2 for their parasitism.The experiment was performed in 5 cm diameter petri plates filled with 1.5% water agar.Five hundred micro-litre (500 µl) drop containing 100 eggs and 50 freshly hatched juveniles of M. incognita was spread on media plate in laminar flow cabinet.Plates were incubated at 25 °C for 15 days.Sterile distilled water containing eggs or juviniles were used as control (Freire & Bridge, 1985;De Leij & Kerry, 1991).Each treatment was replicated six times.Eggs and juveniles were stained with acid fuchsin (3.5 gm acid fuchsin in 250 ml of acetic acid and 750 ml of distilled water) and then examined under the microscope.Number of infected and uninfected juviniles and eggs were counted (Kerry & Crump, 1977).

Root Coating With Mycelia of P. chlamydosporia
Plugs (4-5 cm) of 10 days old culture were transferred in to erlenmeyer flasks containing potato dextrose broth and then incubated in shaking incubator at 170 rpm for 10 days at 25 °C.Mycelial mat was cut into fragments with a blender and poured in beaker.Roots of tomato seedlings were dipped in the beaker and hand shaken for 5-10 seconds and then planted in pots and field.

Root Coating With Chlamydospores of P. chlamydosporia
Wet roots of tomato seedlings were dipped in the bottles containing chlamydospores developed on millet and hand shaken for 10-20 seconds so that the spores adhere to the roots.The seedlings were transplanted to pots and field.

Screen House Study
Seedlings of tomato germplasm (Money Maker) was grown in earthen pots in screen house.Seedling of three weeks old were planted in plastic pots filled with autoclaved soil (2.5 kg) with sand, silt and clay contents of 2:1:1.Seven days after trasplantation, 10,000 fresh eggs of M. incognita were applied to rhizosphere of each plant.Small holes around the plants were made and eggs and biocontrol agent was applied according to the treatments.The experiment was performed with fifteen treatment and five replication arranged in randomized complete block design (RCBD) (Table 1).The experiment was terminated after 60 days of inoculation.Data were noted for shoot length (cm), fresh and dry shoot weight (g), fresh and dry root weight (g), number of galls and number of eggs per plant root system, numbers of flowers and fruits per plant, and fruits weight per plant (kg).

Field Experiment
Field study was conducted in a root-knot nematode infested field in the two crop seasons i.e., summer and winter crop.Tomato cv.Rio-grande was used in both seasons.Twelve (12) treatments were applied to four week old seedlings in randomized complete block design and 4 replications.Each replication consisted of 10 plants.Cross contamination between treatments was prevented by following the practices suggested by (Verdejo-Lucas, Sorribas, Ornat, & Galeano, 2003).Treatments applied in field experiment were listed in Table 2. Field experiment was ended at 50 days after transplantation.Data regarding different parameters noted in sreen house trial was also recorded in field trial.Furthere more initial nematode population (Pi) and final nematode population (Pf) were also measured in field trial.

Stastical Analysis
The data collected from field, green house and experiments were subjected to analysis of variance (ANOVA) using MSTAT-C (Statistical software package).Least significant difference test (LSD) test was applied to the data for the separation of means at P ≤ 0.05 (Steel &Torrie, 1980).
Significant increase was noted in plant height (cm) (37.6%), fresh shoot wt (g) (55.4%) and dry shoot wt (g) (29.3%) as compared to control under screen house conditions (Table 5).Fresh root weight (g), dry root weight (g), number of galls per plant and number of egg masses per gram of galled tissue of root were decreased by 22.1%, 47.7%, and 80.8% and 53.0%respectively.Number of flowers and tomato yield in terms of numbers of fruits and total fruit weight were also increased in treated pots.In summer field trials, data indicated that no significant difference was observed among treatments (Table 5).However, considerable effect of the fungal applications was seen in the winter crop (Table 6).T 6 was most effective than other formulations.Plant height (44.3%), fresh shoot wt (g) (51.0%) and dry shoot wt (g) (70.3%) were increased as compared to control.Fresh root weight (g) (38.6%), dry root weight (g) (35.5%), number of galls per plant (54.9%) and number of egg masses per gram of galled tissue of root (66.3%) were effectively reduced.Significant increase occurred in number of flowers (51.0%), fruits (34.6%) and total fruit weight (62.1%).The Root-knot nematode final population (Pf) of winter crop significantly decreased in all the treatments.Maximum decrease in final population was recorded in T 6 (46.9%).
Current study showed that culture filtrate of P. chlamydosporia isolates strongly affected J2 mortality and egg hatching of M. incognita.We also noted that increasing the concentration of culture filtrate increased egg hatch inhibition and J 2 mortality, and thus confirming the previous finding (Mukhtar & Pervaz, 2003).Effect of the CF from several fungi on egg hatch and J 2 mortality of nematodes have also been previously reported (Randhawa, Singh, Sandhu, & Bhatia, 2001;Zaki, 1999).Isolates PC-6 and PC-1 strongly inhibited egg hatch and killed J 2 .Both isolates (PC-1 and PC6) showed considerable variations in egg parasitism.PC-1 parasitized maximum number of eggs (73.7%) followed by PC-6 (68.3%).Ebadi et al. (2009) also recorded more than 85% of M. javanica eggs parasitized by P. chlamydosporia.
Non-significant difference was noted between PC-1 and PC-6 in the case of juvenile mortality.On the other hand PC-1 seems to be more effective than PC-6 in killing nematode eggs.This may be due the high production of egg shell degrading enzymes by PC-1.Nematicidal effect was not seen in potato dextrose broth (culture media) and thus did not differ significantly as compared to water (control).This supports the finding of Singha and Mathurb (2010) and contradicts the findings of Nitao et al. (1999).
The selected isolates (PC-1 and PC-6) of P. chlamydosporia were tested under screen house and field conditions using different application rates and methods.Inoculum level of the bio agent has a significant effect on its host (Zareen & Zaki, 2001).Both PC-1 and PC-6 were found to be more active when applied at rate of 6 × 10 3 chlamydospores/gm of soil.Significant difference was noted between treatments where chlamydospores applied at rate of 2 × 10 3 and 6 × 10 3 per gram of soil.Both isolates of P. chlamydosporia suppressed nematodes and improved plant growth in tomatoes, however, no statistical difference was recorded between the isolates.Under field conditions, isolates did not exhibit any significant difference for their effectiveness in summer crop.On the other hand, significant variation was noted among treatments in winter crop.Galling indices was highly reduced in winter crop irrespective to the application rate and method of application as compared to summer crop.This might be due to low cfu/gm of both isolates in soil during summer and thus confirming the previous finding (Vieira dos Santos, Esteves, Kerry, & Abrantes, 2014).

Conclusion
The isolates of P.chamydosporia produced compounds which have potential as novel nematicides.In the present study, the two isloates (PC-1 and PC-6) were effective against egg hatching, egg parasitism mortality of second stage juveniles of M. incognita under invitro condition.In screen house and field trial both isolates showed good biocontrol potential against M. incognita.Root coating with chlamydospores application method was found to be more effective than mycelial root coating in the current study.No lesions were noticed on P. chlamydosporia treated roots of tomato plant which reveals that the tested fungus is safe.Further research work is needed for the molecular characterization of these isolates, and isolattion and structure elucidation of nematicidal compounds produced by two isolates.

Table 1 .
List of treatment applied to tomato germplasm under screen house conditions

Table 5 .
Evaluation of P. chlamydosporia for controlling Meloidogyne incognita in summer crop under field conditions

Table 6 .
Evaluation of P.chlamydosporia for controlling Meloidogyne incognita in winter crop under field conditions