Isolation, Molecular Identification and under Lab Evaluation of the Entomopathogenic Fungi M. anisopliae and B. bassiana against the Red Palm Weevil R. ferrugineus in Gaza Strip

Plant diseases generate challenging problems in commercial, agriculture and pose real economic threats to both conventional and organic farming systems. The red palm weevil (Rhynchophorus ferrugineus) (RPW) is one of the most destructive pests of palms in the world. Nowadays, control methods revolve around treatments based on chemicals, biotechnological systems using semi-chemicals or the development of the sterile insect technique (hardly sustainable at this time) and biological control. Biological control as the use of natural microorganisms, extracted products from microorganisms or genetically improved to resist or eliminate of pathogens. Our aim was to evaluate the entomopathogenicity of indigenous Beauveria bassiana and Metarhizium anisopliae obtained in Gaza strip against larvae and adults of R. ferrugineus in order to identify indigenous strains potentially suitable for Red Palm Weevil biological control. B. bassiana & M. anisopliae were isolated from larvae and adult dead of RPW from different position of Gaza strip. Morphological analysis of the isolated fungi and molecular identification was determined using PCR technique. Also, the efficiency of the isolated fungi were evaluated under lab conditions and optimized as a biological agent. On the anther hand, the ability of treated RPW male to infect females is examined and calculated using Abbott's formula. Our results showed that the B. bassiana and M. anisopliae exhibited a good biological control agent against larvae and adults of RPW. The pathogenicity of the two most virulent isolates and the toxicity assay on larvae showed a highest mortality percentage nearly to 100% by 6 days after spraying the larvae with 3.4 × 10 spores/ml of B. bassiana. The mortality percentage reaches to 90% after spraying the larvae with 3.6 × 10 spores/ml of M. anisopliae. The mortality for the adults treated with pesticide arrives to 50% and the control group 10% at the same time. The results revealed that the infection of the adult males by Entomopathogenic fungi (EPF) can be disseminated into the healthy population, after RPW treatment with B. bassiana and M. anisopliae. How to cite this paper: El Kichaoui, A.Y., Abu Asaker, B.A. and El-Hindi, M.W. (2017) Isolation, Molecular Identification and under Lab Evaluation of the Entomopathogenic Fungi M. anisopliae and B. bassiana against the Red Palm Weevil R. ferrugineus in Gaza Strip. Advances in Microbiology, 7, 109-124. http://dx.doi.org/10.4236/aim.2017.71009 Received: December 12, 2016 Accepted: January 17, 2017 Published: January 20, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

The red palm weevil R. ferrugineus is one of the most destructive pests of palms in the world. This weevil affects more than 20 palm species (Barranco, et al. 2000) [4] including the date palm. R. ferrugineus was introduced in Spain mainland in 1995 ( Barranco, et al. 1996) [5], and then spread to all palm growing areas in the Mediterranean and recently also to the Canary Islands. The pest has caused large economic losses in date palms worldwide for the last 30 years (Murphy and Briscoe, 1999;Faleiro, 2006; Güerri-Agulló, 2010) [6] [7] [8].
Biological control as the use of natural microorganisms, extracted products from microorganisms or genetically improved to resist or eliminate of pathogens. It is performed by using microorganisms from the environment itself directly or makes some changes in their properties, to increase their effectiveness or use one of their products.
Advantages of using of this method are to reduce the costs of pest control. Additionally, it preserves human health and environment from pollution, of chemical pesticides usage. Many researches and studies concern only on the use of pathogens such as entomopathogenic nematodes, bacteria and EPF in controlling RPW. Naturally occurring bio-control agents are alternative tools to the use of hazardous synthetic insecticides.
The present study aim was to evaluate the entomopathogenicity of indigenous Beauveria bassiana and Metarhizium anisopliae obtained in Gaza strip against larvae and adults of R. ferrugineus in order to identify indigenous strains potentially suitable for Red Palm Weevil biological control.

Purification of Fungi by Using Selective Medium
Selective medium is generally required for isolation of B. bassiana and M. aneosiplaia from soil. Sterilized DOC2 medium of B. bassiana and Oatmael agar medium (OMA) of M. aneosiplaia poured into 15 cm petri dishes (Liu, et al. 2015) [12]. The quantity of B. bassiana and M. aneosiplaia was significantly increased using Potato dextrose agar (PDA) and incubated at 25˚C in the total darkness.

Spore Suspension
Liquid medium (PDB) was used for production of spores required for experiments. Liquid mediums were autoclaved and inoculated with fungal spores propagated on PDA.
Spores were harvested from 2 -3 weeks old surface cultures by scraping and used to inoculate the liquid medium in flasks and then flasks were held on a shaker (110 rpm) for 5 days at 25˚C. The suspensions were stirred and filtered through a single layer of linen to remove culture debris and mycelia. After this time, the blast spore concentrations were determined using a haemocytometer and were calibrated to 3.4 × 10 8 spores/ml for B. bassiana and 3.6 × 10 8 spores/ml of M. aneosiplaia respectively. These suspensions represented the primary stock suspensions to making the spore product (Gindin, et al. 2006) [13].

Morphological Identification of Fungal Isolates
When fungal colonies sporulated on PDA, small plaques from the edge and the center of each growing colony were transferred onto glass slides, and then were examined using a compound light microscope, for characteristics of their vegetative and reproductive structures such as hyphal color and structures, shape and size of conidia and conidiophores (Yu, 2010) [14].

Specific DNA Detection of M. anisopliae and B. bassiana by PCR
The nuclear rDNA region spanning the ITS1, ITS2, 5.8S rRNA gene for isolated fungi additionally the SCAR fragment for B. bassiana. All genes were amplified by polymerase chain reaction (PCR) from tow strain and all primers were presented in Table 1.
A. Y. El kichaoui et al.

Bioassay (Contact Application of Fungi)
Evaluation On the other hand, treatment the male adults to evaluate if the male infect the female by fungi or not, after divided all insects into 4 groups as mentioned above.

Development of the B. bassiana and M. aneosiplaia Based Bio-Fungicide as a Stable Liquid Formulation
The most effective B. bassiana and M. aneosiplaia isolate will be maintained in appropriate conditions and heavily tested to give a stable liquid formulation of product based bio-fungicide.

Data Collection and Statistical Analysis
The data was subjected to statistical analysis, virulence was expressed by cumulative mortality (%), treatment efficacy (Abbott's Formula) (ABBOTT, 1925) [20], within 28 days after treatment. The bar chart tested by using SPSS Statistics 17.0 (SPSS Inc., 2009).

Isolation of B. bassiana & M. aneosiplaia Fungi
B. bassiana and M. aneosiplaia was isolated from dead larvae and adult of RPW, which presented in Figure 1.

Cultural Characterization & Microscopic Examination for B. bassiana & M. aneosiplaia Fungi
According to the macroscopic examination for M. aneosiplaia, we found one distinct strain based on the differences in colony morphology. After 10 days of incubation, the culture produces a white mycelial margin with clumps of more or less vermiculate branching conidiophores. The colors vary from olivaceous buff to cream color to dark green ( Figure 2). This is akin to the observations of (Bridge, et al. 1993) [21]. However, there were founded the conidial shape and size of the two kinds of isolates: cylindrical with obtuse ends, slightly narrowing in the center, and the conidial width (1.5 to 3 μm) and length (4 to 8 μm), which was presented in Figure 3.
Generally, in culture, B. bassiana grows as a white mould. It produces many dry, powdery conidia in distinctive white spore balls. Each spore ball is composed of a cluster of conidiogenous cells. This result supported by (Elkichaoui, et al. 2016) [22].
Microscopic observation results showed that hyphae size about 1 -2 μm which grouped on conidiogene cells with 3 -6 μm in size. Hyphae then branched and formed conidiogene cells with bottle like form, small neck, and branch long were up to more than 20 μm and 1 μm wide, which illustrated in Figure 4.
Fertile hyphae was found on branch, circular and normally thicken or swollen. While

Enrichment for Tow Fungi and Spore Suspension
Liquid medium Potato-dextrose-broth (PDB) was used for production of spores re-

PCR Amplification of β-Tubulin
Part of β-tubulin gene was amplified successfully for tow fungal species. The size of this part in B. bassiana isolate was found to be greater than the corresponding one in M. anisopliae isolates (500 bp and 380 bp), respectively, Figure 6(e). β-tubulin was developed for sequencing purposes as described by (Bischoff, et al. 2006) [26].

PCR Amplification of SCAR Region for B. bassiana
Standard PCR examines utilizing primers GHTqF1 and GHTqR1 against strain of B. bassiana created a 96 bp, recouped from tainted RPW grown-ups taking after shower application. The PCR item produced was of the anticipated length in view of the SCAR part whereupon the preliminaries were based. The fragment sizes of SCAR regions, as obtained by gel electrophoresis, were presented in Figure 6.

Pathogenicity of Entomopathogenic Fungi to R. ferrugineus Eggs
The pathogenicity of the two most virulent isolates of M. anisopliae and B. bassiana, selected in the initial screening on adult and larvae, was tested against R. ferrugineus eggs. Both isolates killed the eggs quickly during 3 days, without preliminary colonization on the egg surface. The characteristic symptoms, which appeared on treated eggs, e.g. loss of, tumefy lethargy and darkening of the eggs, appeared 2 -3 days after treatment; subsequently the eggs were destroyed and disappeared in the substrate.

Pathogenicity of Entomopathogenic Fungi to R. ferrugineus Larvae
The larval mortality was measured by Bottle equation. Significant difference in growth

Pathogenicity of Entomopathogenic Fungi to R. ferrugineus Adult
Results of the first experiment indicated that the mortality of R. ferrugineus adults differed according to the fungus application method. The mortality of adult weevils was recorded for 28 days after contact with spraying with spore suspension, which presented the adult were treated by 3.4 × 10 8 spores/ml of B. bassiana and 3.6 × 10 8 spores/ml of M. anisopliae. The maximum mortality of weevils reached 95% by 28 days after spraying with B. bassiana, but 86.6% after spraying with M. anisopliae at the same time and was supported by recent study for Francardi, 2012 [27], when studying the effect of M. anisopliae was isolated from Italy, was appeared also influenced by the type of infecting substratum as resulted higher on larvae (100% mortality) and adults (90% mortality).
Mortality in control groups (aqueous D.W) was 0% and 10%, but the maximum mortality of RPW reached 50% by 28 day after treated with chemical pesticide (Figure 8).

Pathogenicity of Entomopathogenic Fungi to R. ferrugineus Adult Male
The results shown in Figure 9 demonstrated that all entomopathogenic fungal strains A. Y. El kichaoui et al.

Discussion
This study provides general information about the isolation and genetic diversity of  (Hall & Papierok, 1982) [35].
The arrival of entomopathogenic fungi to infest the host is through the cuticle is considered successfully control of RPW, which that involves complex biochemical interactions between the host and the pathogen (fungus) such as B. bassiana & M. anisopliae before germination, penetration, growth, and reproduction of the fungus. Before to host invasion, there are certain characteristics of fungi that designate them virulent or avirulent strains. So, there is a constant struggle between host and pathogen that ultimately lead to the success or failure of pathogens.
In case of compatible interaction, the pathogen must have high number of conidia with strong adhesion that ultimately penetrate into the host through directly penetrating structures. Moreover, the invading pathogen must have the capacity to bypass or overcome the host immune system by producing toxins (Hussain, 2013) [36].
The mating behavior of the RPW resulted in transferring the spores from infected male insects to the uninfected female insects. The pathogenic efficacy of B. bassiana & M. anisopliae indirectly infect females. The efficacy of indirect infection was high. The period required to obtain the maximum mortality of 90% was extended to compare the directly infected females or males, but the maximum mortality of 20% for male adult was treated by chemical pesticide. However, the time to death of indirectly infected females from mating with the infected male insects ranged from 3 to 6 day after mating.
These results supported by Hajjar, et al. 2015 [37]. The pathogenic efficacy of the indirect infection was high, as all the indirectly infected insects of both sexes were killed after 3 d of mating. The early deaths of the directly infected insects could be due to the high load of B. bassiana spores on the body as compared with indirectly infected red palm.
Recently (Llacer, et al. 2012) [38], advanced the possibility of using sterile irradiated males as a vector of B. bassiana for microbiological control of R. ferrugineus. In laboratory bioassays, the transmission system successfully attracted, infected and released weevil adults after they contacted cereal substrata inoculated with indigenous strains of B. bassiana and M. anisopliae (Francardi, 2012) [27].
Results indicate that the EPF against R. ferrugineus can also provide an excellent alternative to chemical control. The EPF of B. bassiana and M. anisopliae strains obtained from different sources. All of the screened EPF strains exhibited pathogenicity to larvae stages of RPW, causing up to 90% -100% mortality of larvae weevils under laboratory conditions. The fungal spores had a significantly reduced survival in comparison with larvae, which emerged in the control treatment.
The effects of B. bassiana treatments on activity and survival of larvae correlate well with previous studies conducted outside of hidden environments (Fargues,  In other side the bioassays, significantly higher mortalities of RPW adult were observed at use of B. bassiana as biocontrol agent against the R. ferrugineus. After spraying RPW adult with spore suspensions (3.4 × 10 8 ml −1 ) of B. bassiana isolates from dead adult, but in the second term the M. anisopliae caused the death for RPW and the mortality percentage 86.6% after treatment the R. ferrugineus adult with spore suspensions (3.6 × 10 8 ml −1 ). While the lower mortalities of RPW adult were observed at use of chemical pesticide at the same time.
In the current bioassays, maximum number of sporulation and mycosis in cadavers of R. ferrugineus was recorded at the highest and sole concentration of B. bassiana & M. anisopliae as compared to all other treatments, after treatment of adult RPW males with EPF. Similar trend in mycosis and sporulation were reported by Riasat, et al. 2001 [41], in (Ramakrishnan, 1999) [42].
Efficacies up to 90% were obtained compare with chemical pesticide 35%, and these results are indicative that contact infection of adults actually occurred and confirm the potential of this strain as a biological control agent against R. ferrugineus. Consequently, adults should be considered as the targets of any treatment involving this entomopathogenic fungus because they are actually the only free-living stage.
Overall, we can see a clear upward trend to use the EPF, which aimed at attracting and infecting adult weevils could prove the most effective way to spread the disease, and this is one of the works that our group is developing at this moments.

Conclusion
This work as a part of general project that aims to solve some of environmental and health problems by reducing the use of chemical fertilizers, pesticides and drugs and replace them by natural material or organisms. B. bassiana & M. anisopliae isolates from the adult RPW used in laboratory bioassays caused high mortality in larvae and adult. Natural infections by B. bassiana and other entomopathogenic fungi found were very high, considering the instar. For these reasons, the use of entomopathogenic fungi can be considered to be useful as a preventive tool in Gaza strip palm protection.
Moreover, the high mortality of treated adults suggests that their use as vectors of B. bassiana & M. anisopliae can represent a potential tool for reducing R. ferrugineus populations in Gaza strip.