WOS: 000400123900016Plant parasitic nematodes such as root-knot nematodes (RKNs) are a major threat to vegetables, fruits and ornamental plants which cause yield loss. One of the best alternative methods of nematode control is using the entomopathogenic fungi (EPF). The aim of this study was to evaluate the effects of two Turkish isolates of entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. (F-56 and F-63), against naturally infected mixed populations of Meloidogyne incognita and Meloidogyne javanica on tomatoes under natural conditions. The experiment contained 3 conidial suspensions (10(6), 10(7) and 10(8) cfu ml(-1)) with 4 replicates of each treatment. A total of 32 plants were arranged in a randomized block. EPF were applied 4 times (15 days before planting, planting, 15, and 30 days after planting). Nematicide [Paecilomyces lilacinus strain 251 (BioAct (R))] was used as a positive control. At the end of the growing season, the efficiency of the applications was determined according to the root gall index (I) and yields of tomatoes (II). The fungi concentration of 10(8) cfu ml(-1) was significantly greater than in the others. This concentration from both isolates [F-56 (4.59 +/- 0.54) and F-63 (2.77 +/- 0.59)] controlled M. incognita and M. javanica and increased the yield of tomatoes. The results demonstrated the suitability of using B. bassiana isolate F-56 and F-63 for reducing RKNs on tomatoes under greenhouse conditions

Kepenekci, I.

Oksal, E.

Saglam, H. D.

Yanar, D.

Yanar, Y.

Kırşehir Ahi Evran University Institutional Repository

Egyptian Journal of Biological Pest Control, 27(1), 2017, 117-120 Nematicidal Activity of Beauveria bassiana (Bals.-Criv.) Vuill. Against Root-Knot Nematodes on Tomato Grown under Natural Conditions  Kepenekci1 ; H. D. Saglam2; E. Oksal3; D. Yanar1 and Y. Yanar1 1  Turkey. 2 Ahi Evran Univ., Dept. of Plant Protection, Fac. of Agriculture  Turkey. 3 Dept. of Plant Protection, Fac. of Agriculture, 44000, Malatya, Turkey.  (Received: February 4, 2017 and Accepted: March 14, 2017)   ABSTRACT  Plant parasitic nematodes such as root-knot nematodes (RKNs) are a major threat to vegetables, fruits and ornamental plants which cause yield loss. One of the best alternative methods of nematode control is using the entomopathogenic fungi (EPF). The aim of this study was to evaluate the effects of two Turkish isolates of entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. (F-56 and F-63), against naturally infected mixed populations of Meloidogyne incognita and Meloidogyne javanica on tomatoes under natural conditions. The experiment contained 3 conidial suspensions (106, 107 and 108 cfu  ml-1) with 4 replicates of each treatment. A total of 32 plants were arranged in a randomized block. EPF were applied 4 times (15 days before planting, planting, 15, and 30 days after planting). Nematicide [Paecilomyces lilacinus strain 251 (BioAct®)] was used as a positive control. At the end of the growing season, the efficiency of the applications was determined according to the root gall index (I) and yields of tomatoes (II). The fungi concentration of 108 cfu ml-1 was significantly greater than in the others. This concentration from both isolates [F-56 (4.59±0.54) and F-63 (2.77±0.59)] controlled M. incognita and M. javanica and increased the yield of tomatoes. The results demonstrated the suitability of using B. bassiana isolate F-56 and F-63 for reducing RKNs on tomatoes under greenhouse conditions.   Key words: Root-knot nematodes, Meloidogyne incognita, M. javanica, Beauveria bassiana, Tomato, Biocontrol.  INTRODUCTION  Plant parasitic nematodes (PPN) usually cause yield loss on many agricultural products around the world. One of the most important groups is the root-knot nematodes (RKNs) (Perry and Moens, 2006 and Kepenekci, 2012). RKNs can be suppressed by chemical control, but its use has been restricted due to its impact on environmental problems, human health and non-target organisms. Therefore, alternative methods are needed to expand effective management strategies for plant parasitic nematodes.  Entomopathogenic fungi (EPF) are among the effective biological control agents (Butt et al., 2001). They are environmentally more acceptable than chemicals (Arici et al., 2012) and also can be used when pests have developed resistance to chemical pesticides (Butt, 2002). Some fungi have shown promising results as a biocontrol agent against the destructive RKNs (Jatala et al., 1979; Stirling, 1991 and Tigrano-Milani et al., 1995). More than 30 genera and 80 species such as Arthrobotrys spp., Fusarium spp., Aspergillus spp., Penicillium spp., Purpureocillium lilacinum, and Verticillium chlamydosporium have been reported (Viaene and Abawi, 2000; Sun et al., 2006 and Bakr et al., 2014).  Beauveria bassiana (Bals.-Criv.) Vuill. is a well known biopesticide for insects, but few studies have done on PPN. B. bassiana grows saprotrophically within the host and produced metabolites such as; beauvericin, bassianin, beauverolides, bassianolide and oosporein. This fungus invades the insect body and its toxins act as an insect platelet aggregation inhibitor (Butt et al., 2001).   The aim of this study was to evaluate two Turkish isolates of EPF, B. bassiana F-56 and B. bassiana  F-63 on the mixed population of Meloidogyne incognita and M. javanica under greenhouse conditions.   MATERIALS AND METHODS  Nematodes A greenhouse (about one and half decares), planted with tomato naturally contaminated with the root-knot nematodes, M. incognita and M. javanica in was selected. New tomato plants, infected with knots, were washed carefully to obtain occupant RKNs on the roots in the greenhouse. After examination of the root galls, abundant mature females were found attached to the roots. On the basis of the perennial pattern of mature females (Southey, 1986), M. incognita and M. javanica were identified (Jepson, 1987 and Eisenback and Triantaphyllou, 1991).  Fungus isolates Cultures of the Turkish isolates, EPFs, B. bassiana F-56 and B. bassiana F-63, were provided by the 118   University, Faculty of Agriculture, Department of Plant Protection, Tokat, Turkey via the Project of GOU-BAP (Project No: 2012/25) from stored at 4oC on Potato Dextrose Agar (PDA) and at 80oC. The fungi were subcultured on PDA (Difco TM, Becton Dickinson and Company, USA) with the help of a sterilized bacteriological loop. The dishes were closed by a parafilm at 25±1°C for 7-14 days. The conidia were harvested, using a sterilized rubber loop, attached to 1 ml borosilicate pipette at the angle of 45°. Scraped material was shifted into sterilized Petri dishes and stored at 4°C in refrigerator. The harvested fungal conidia were incorporated to sterile 0.05% Tween-80 solution and the material was stirred for complete homogeneity (Wakil et al., 2012). Serial dilutions were prepared and the number of conidia was measured by a haemocytometer  to achieve the concentrations of 106, 107 and 108cfu ml-1.  Greenhouse experiments The experiment was conducted between the 5th of August 2014 and the 8th of January 2015. Ilgin  F1-tomatoe (Solanum lycopersicum) was used as a susceptible variety to RKN. BioAct® (nematicide based on entomopathogenic fungus, Paecilomyces lilacinus strain 251) was used as a positive control. Only water was applied as a negative control via drip irrigation. The experiment contained 3 different conidial concentrations (1×106, 1×107 and 1×108cfu ml-1). One parcel had 2 rows and each row contained 16 plants, totaling 32 plants were arranged  (8 m×1. 5 m =12 m2) in a randomized block design. Four replicates of each treatment were performed. The 2.5 m safety lanes were left between parcels. Entomopathogenic fungi (EPF) were applied 4 times: 15 days before planting, planting, 15 and 30 days after planting, with a watering pot (the extracts were applied 1 ml plant-1). After application, drip irrigation was opened approximately 1 hr for diffusing the bio pesticide in the soil. The parameter of gall index (root knot), yield (kg plant-1) and the effect (%) were evaluated. Temperature and relative humidity were recorded by HOBO [15.57-43.12ºC (24.85ºC ±6.88) and R.H.% of 67.33-96.97 (86.24±8.70%)] during the experiment. To evaluate the effect of treatments on yield, mature (red color) tomatoes were harvested regularly. The first fruits were harvested on the 12th of September 2014 and completed on the 8th of January 2015. Twenty tomato plants were selected randomly from each treatment and harvested tomatoes were weighed and recorded for each plant for the entire season (8 to 10 times). At the end of the season, mature tomatoes on the plants were collected and plants were uprooted the same day. The damage caused by RKN was evaluated according to Zeck galling scale. Twenty plants were selected randomly from each plot and galling damage was rated on a  0-to-10 scale, as 0= no galls, 1= very few small galls, 2= numerous small galls, 3= numerous small galls of which some were grown together, 4= numerous small and some big galls, 5= 25% of roots severely galled, 6= 50% of roots severely galled, 7= 75% of roots severely galled, 8= no healthy roots but plant was still green, 9= roots rotting and plant dying, and 10= plant and roots dead (Zeck, 1971).  Statistical analysis All data were analyzed using ANOVA by SPSS package program. The mean separations were (SPSS, 1999).  RESULTS AND DISCUSSION  The results showed that the concentration of 1×108 cfu ml-1 of B. bassiana F-56 and  F-63 were found more effective than other concentrations (1×107 and 1×106). The highest effect on gall index was recorded on the concentration of 1×108 cfu ml-1of B. bassiana F-63 (2.77±0.59) and found statistically the same group with positive control (1.34±0.35). Another effective application was found in the concentration of 1×108 cfu ml-1 of B. bassiana F-56 (4.58±0.54). Negative control  recorded the highest gall index value (8.66±0.45) compared to the others (F= 19.57; df: 7.31; P<0.05) (Fig. 1). The concentration of 1×108cfu ml-1 of B. bassiana F-63 (66.60±9.25%) showed the highest effect on gall index compared to the other concentrations. The highest yield was demonstrated by B. bassiana F-56 and F-63 (5.00±0.09 and 4.95±0.31 Kg plant-1, respectively). The lowest yield was found on negative control parcels (3.17±0.69 Kg plant-1); while the highest yield (62.04±28.88) was obtained at the concentration of 1×108 cfu ml-1 of B. bassiana F-56, followed by the concentration of 1×108 cfu ml-1 of B. bassiana F-63 (61.02±33.82%). The lowest effects were recorded at the lowest concentrations of both B. bassiana F-63 and F-56 (Fig. 1). It was found that the most virulence of the EPF depends on increasing the concentration.  Entomopathogenic fungi are one of the effective methods to control the plant parasitic nematodes (Butt et al., 2001). Khalil et al. (2012) reported that  P. lilacinus [Purpureocillium lilacinum (syn: Paecilomyces lilacinus)] was the most effective treatment against M. incognita, a pot experiment under greenhouse conditions, recorded 88.23% gall and 76.94% egg mass reduction. Zakaria et al. (2013) found that Verticillium chlamydosporium and symbiotic bacterium Photorhabdus luminescens were alone or combined or with soil amendment  119Fig. (1): Effect of different concentrations of Beauveria bassiana on Meloidogyne incognitaand M. javanica infection of tomato plants (A) root gall index and yield, (B) percentage of efficacy of B. bassiana. Capital (yield) and small (root gall index) letters denote within the column.significantly reduced nematode infections. A few studies have been done on the effect of B. bassianaon the plant parasitic nematodes. Ekanayake and Jayasundara, (1994) found that P. lilacinus was more effective than B. bassiana as toxin beauvericin produced from B. bassiana showed weak nematicidal activity against M. incognita. Chen et al. (1996) evaluated 21 isolates of 18 fungal species and found that B. bassiana had less parasitism of nematode eggs, but reduced hatching of Heterodera glycines in comparison to P. lilacinus that found moderately pathogenic to the egg and reduction of hatching. Sun et al. (2006) determined microflora on egg and females of Meloidogyne spp. and 455 fungal isolates were obtained. The predominant fungal species observed was P. lilacinus (49.3% of the isolates) and B. bassiana was found only in one location. Most of the isolates were collected from Meloidogyne spp. eggs. In vitro study showed that B. bassiana parasitized (100%) of the eggs, the egg hatching rate was (36%) and juvenile mortality rate was (18.1%) of M. hapla. Liu et al. (2008) evaluated culture filtrate of isolate B. bassianaagainst eggs and juveniles of M. hapla in vitro and glass-house conditions. They found the filtrate of B. bassiana successfully inhibited the invasion of M. hapla juveniles and 1× dilution of B. bassiana isolate showed better inhibition (98.61%) rate compared with the chemical pesticide of Aldicarb (88.89%). Although the 1× dilution of B. bassiana isolate had showed the highest effect on M. hapla, also inhibited tomato plant growth. 5× dilutions of B. bassianaisolate inhibited nematodes like Aldicarb, but also better plant growth were recorded. Bustos et al. (2014) evaluated a crude filtrate from the strain of Purpureocillium sp. UdeA0106 against M. incognitaand M. javanica in in vitro conditions and found the full strength filtering (100%) was the most effective in inhibiting both egg hatching and juvenile mobility.As a conclusion, B. bassiana showed that the concentration (108cfu ml-1) of F-56 and F-63 had a promised nematicidal activity of root-knot nematodes as well a positive effect on yield. This result indicated that applying the biological control agent inoculum level was important for biocontrol efficacy. These native isolates of B. bassiana could be suggested as biological control agents of Meloidogyne spp.ACKNOWLEDGMENTTurkey) who permited us to work in his greenhouse and for his help during all the greenhouse studies.REFERENCESArici, S. E., I., Gülmez, H. Demirekin, H. entomopathogenic fungus Fusarium subglutinansagainst Aphis fabae Scopoli (Hemiptera: Aphididae). Turkish Journal of Biologial Control, 3:89-96.Bakr, R. A., M. E. Mahdy and E. M. Mousa. 2014. 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Nematicidal Activity of Beauveria bassiana (Bals.-Criv.) Vuill. against Root-Knot Nematodes on Tomato Grown under Natural Conditions

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