USING Rhabditis blumi SUDHAUS AS BIOLOGICAL AGENTS TO CONTROL THE PALP BORER, ARABIAN RHINOCEROS BEETLE, Oryctes agamemnon arabicus

This study was aimed to use pathogenicity of entomopathogenic nematodes (EPN) Rhabditis blumi Sudhaus (Nematoda: Rhabditida) against palm borer Arabian Rhinoceros Beetle (ARB), Oryctes agamemnon arabicus was evaluated in the lab. And date palm orchards during 2015 2017. In the lab tests, EPN was used against the larvae and adults as a direct spray and as treated food (pieces of fresh tissue of the frond bases) at a rate of 0, 500, 1000, 1500 Infective Juveniles (IJs) per mL of R. blumi. Results indicated that EPN caused 89%, 61% and 25%, 20% mortality when used as a direct spray and as treated food on larvae and adults of ARB, respectively. Results of field experiments showed that injection of 50 mL per palm tree with a concentration of 1500 IJs/mL of R. blumi inflected about 45.5% mortality in ARB larvae infesting the tree. Meanwhile, the Population density of ARB larvae reduced to 45.8%, 59.6% during the first and second year of treated date palm trees by injection method respectively. The results of this investigation illustrate the possibility of using EPN R. blumi as a biocontrol agent for managing borers in date palm orchards under field conditions.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
Oryctes agamemnon arabicus causes severe damages to the bases of fronds and bunches making long tunnels inside the tissue, which are acting as weakening and breaking factors for these parts (13). Many control methods have been used through different application methods: spray, drench and injection (5, 88, and 11) against to date palm pests. Khalaf (14) Reported that the application of thiamethoxam and imidacloprid against ARB larvae resulted in 85.8%, 100% mortality in injection method compared with 75%, 80% in the drench method respectively.Khudair (15) Tested locally isolated entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against ARB larvae and reported a high mortality rate among larvae reaching up to100% after 29 days of treatment under lab conditions. Entomopathogenic nematodes will only be widely used as pest control products when they become available on demand by the different clients, commercial growers and small farmers (16). Rhabditid nematodes are generally recognized as bacterivores and often associated with invertebrates, their relationship with invertebrates is known as necromancy, which means waiting for the death host (6, 20, 23, 28 and 26). Khalaf (14) Tested entomopathogenic nematodes (EPN), Rhabditis blumi, and the entomopathogenic fungi (EPF), Beauveria bassiana as biocontrol agents against larvae and adults of ARB, O. agamemnon arabicus. Biological control potentials of R. blumi against 5 coleopteran species and 5 lepidopteran species was evaluated by Park (19). This study aimed to investigate the efficacy of EPN, Rhabditis blumi as entophytic biocontrol agents against palm borers, Oryctes spp. especially ARB, O. agamemnon arabicus under laboratory and field conditions. Another objective was to investigate the feasibility of application and persistent in date palm orchards.

MATERIALS AND METHODS Borers and nematodes cultures:
Samples of ARB O. agamemnon arabicus were obtained from the lab. Colony reared on natural foods (palm tissues, fresh frond bases pieces) at 25±2°C, complete darkness and 65% relative humidity in the IPM Center, Directorate of Agricultural Research, and Ministry of Science and Technology (14). R. blumi was obtained from the lab. Colony reared on larvae of wax moth Galleria mellonella in labs of IPM center (14).

Laboratory experimental treatments
The laboratory trials were conducted in Biological Control Dept. of IPM Center. Laboratory experiments included using four concentrations (0, 500, 1000 and 1500 IJs per mL) of R. blumi as direct spray on larvae and adults of ARB or mixed with their food (pieces of frond bases tissue). Fresh food pieces were added regularly through the period of treatment to keep enough fresh food to the larvae. Five replicates, 6 larvae/rep as larval treatment and four replicates, 5 adults/rep as adult's treatment were used for each treatment as for laboratory experiments. Larval and adults mortalities were counted in all treatments after 24, 72, 96 and 120 hours of treatment.

Field experimental treatments
Field experiments, injection of 50 mL solution of EPN 1500 IJs/mL was done through tree trunk using 50 mL syringes after drilling holes with a brad point drill-bit (diameter, 20mm and length, 200mm) 1m above the ground level ( Fig. 1-A, B). The same number of trees were left as control treatment in each orchard. Five replicates (trees) were used for each treatment. Larvae of ARB in trees crown were collected after four weeks of injection EPN, dead and live larvae were counted in each treatment and kept in plastic containers ( Fig.1-C). Also, fresh frond bases tissues were collected and healthy larvae of G. mellonella were added to test if it contains EPN, R. blumi in each treatment. Samples of tissue were taken after 4 weeks of treatment for examination and to explore the presence and movement of EPN through plant tissue. Larval mortalities that infected by EPN in each treatment were counted. Efficacy and persistent of entomopathogenic nematode Rhabditis blumi in date palm orchards: Efficacy and persistent of EPN R. blumi were studied in two date palm orchards, the first one was untreated (control) orchard, the second orchard was treated with EPN mixed in 50 mL at a concentration of 1500 IJs by used injection methods in ten tree trunk. EPN distribution and efficacy in reduction of ARB Larvae population density were counted by calculating larvae in ten palm tree Brem variety in each orchard before treatment and during the first and second year after treatment.

Experimental design and data analysis
The experimental designs implemented were complete randomized design and randomized complete block design with four replicates . Genstat program was implied in statistical analysis and determine the significances efficacies. Henderson-Tilton, s formula (1955) (10) was used to calculate corrected mortality efficacies% on larval and adults ARB treated with EPN in lab treatments, while Schneider-Orelli, s formula (1981) (10) was applied for measuring efficacies of the EPN tested on ARB at field treatment. Table 1 indicates that the mortality percentage of ARB larvae after direct spray under laboratory conditions with EPN R. blumi revealed that highest concentration (1500 IJs/mL) caused the highest percentage of mortality 89% after 120 hr., while the lower concentrations of 1000 and 500 IJs/mL recorded mortality of 79% and 57% respectively. Meanwhile, mortalities reached 50%, 54% and 61% at concentrations of 500, 1000 and 1500 IJs/mL respectively in experiments when EPN was mixed with larval food (table1).   Adult's mortality for direct spray and treated food were 0%, 15%, 20%, 25% and 0%, 10%, 15%, 20% at concentration 0, 500, 1000 and 1500 IJs per mL respectively (Table 2).  Field efficacy results indicated that the mortality percentage of ARB larvae after trunk injection with 50 mL in concentration 1500 IJs per mL reached 45.5% (calculated in the crown tree only) after 4 weeks of treatment (Table 3). Results of field studies revealed that there was an acceptable efficacy of applying local isolate of EPN R. blumi (isolated locally from Iraqi date palm orchards ecosystem) as biocontrol agents. The EPN R. blumi can persist habitat causing more reduction in the population density of ARB, O. agamemnon arabicus larvae. In addition, results indicated that the EPN, R. blumi solution could translocate through date palm tissue after injection in the trunk. Park (19) Reported that EPN R. blumi against major cruciferous insect pests and evaluated pathogenicity in lab and greenhouse and showed that EPN caused high mortality rate in larvae. Entomopathogenic nematodes have certain advantages over chemical as control agents; it's a non-polluting agent and thus environmentally safe and acceptable (9). There are many reports for rhabditid nematodes causing mortality of various invertebrates' species: beetles, termite, millipede and rice yellow stem borer (1, 7, 22, 23, and 25). (2, 3,11,24 and 27) they found that the Infective Juveniles of rhabditid nematodes enter an invertebrate, remain until it is dead, and complete their development by feeding on bacteria growing inside the cadaver of insect, and it usually possesses some attributes of a potential biological control agents, such as short life cycle, easy culture condition with bacteria, high fecundity and virulence, and good association with invertebrate pathogenic bacteria. EPN Rhabditis blumi showed significant mortality against Arabian rhinoceros beetle Oryctes agamemnon arabicus, but the mortality rate of larvae was higher than that of adults in lab trails at direct spray or treated food. When EPN was mixed with 50 ml of water at a concentration of 1500 IJs/ml and injected in the trunk, a moderate mortality rate was reported among ARB larvae. However, dispersal and efficacy were increased in treated orchards after one year of treatment.