Pathological

Communication

Biosci. Biotech. Res. Comm. 11(3): 393-401 (2018)

Isolation, phenotypic and genotypic characterization

of indigenous

Beauveria bassiana

isolates from date

palm infested with

Rhynchophorus ferrugineus

in Hail

region, Saudi Arabia

Khalid A. Asiry

*, Abdel Moneim E. Sulieman

, Naimah A. Al-Anazi

, Vajid N. Veettil

Mohanad Abdelgadir

and Ibrahim Alkhregi

Department of Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia

Department of Biology, Faculty of Science, University of Hail, Saudi Arabia

ABSTRACT

The red palm weevil (RPW) Rhynchophorus ferrugineus overrun date palm ranches in many parts of Saudi Arabia,

henceforth causes massive economic losses. Integrated pest Management (IPM) of the RPW by utilizing Entomopath-

ogenic fungi (Beauveria bassiana), which has antagonistic activity against many insect pests, was the main objective

of the current study. In the present study, soil samples, samples of dead red palm weevils (RPW) and palm fronds were

collected according to RPW incidence map of Hail region, Saudi Arabia. Isolates of entomopathogenic fungi were

isolated from the dead RPW adults and larvae. The fungal culture (BSA1, BH-2 and BH-3) was preserved and main-

tained for further analysis. Morphological and biochemical characterization of the antagonist fungi were employed

and con rmed that the fungus belonged to Beauveria spp. Further, this fungal isolates were propagated and prepared

for genetic characterization. Sequencing of internal transcribed spacers ( ITS1 and ITS2) region was shown that three

polymorphic ITS regions. The molecular identi cation of the fungus strain was employed at King Faisal University-

the College of Agriculture and Food Science and con rmed that the fungus identi ed as Beauveria bassiana as the

 rst record of this bene cial species in Hail region.

KEY WORDS: DATE PALM, RED PALM WEEVIL, FUNGUS, MORPHOLOGICAL, BIOCHEMICAL, MOLECULAR IDENTIFICATION

393

ARTICLE INFORMATION:

*Corresponding Author: Prof_1974@hotmail.com

Received 17

July, 2018

Accepted after revision 24

Sep, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.3/7

394 ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

BEAUVERIA BASSIANA

ISOLATES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Khalid A. Asiry et al.

INTRODUCTION

The red palm weevil (RPW) Rhynchophorus ferrugineus

Olivier (Coleoptera: Curculionidae), is an overwhelming

palm pest that can cause tremendous  nancial losses

worldwide throughout the previous 30 years, as they are

notable to assault several species (more than 200) of palm

including the date palm (Phoenix dactylifera L.) (Mur-

phy and Briscoe, 1999; Barranco et al., 2000; Faleiro,

2006). These large economic losses in date palms could

ascribed to the way that, to-date, there are no powerful

control measures. Rhynchophorus ferrugineus is broadly

geographically distributed in Africa, Asia, Europe, Oce-

ania, and North America (EPPO, 2006; 2007a; 2007b;

2009; Azmi et al., 2017).

The entomopathogenic fungus Beauveria bassiana

(Ascomycota, Hypocreales) has demonstrated use within

insect biocontrol administrations for concealment of

numerous crop. It infects a an extensive variety of insect

pests of socioeconomic importance pests (Bing & Lewis

1991, 1992, Krueger & Roberts 1997; Mulock & Chan-

dler 2000). Recently, B. bassiana, was additionally found

to occur naturally as an endophyte in plant tissues, for

example, as leaves, twigs, wood and bark in speci c

plants like maize, cotton, wild cacao, white pine, coffee

and furthermore been set up as an endophyte arti cially

in speci c crops like, maize, cotton, tomato, opium

poppy, cacao, coffee, date palm, banana, sorghum and

jute. The entomopathogenic fungus B. bassiana isolated

from dead R. ferrugineus cadavers gave more mortality

compared to the other isolates. In the virulence bioassay

two isolates of B. bassiana shown the highest percentage

of larval and adult mortality at all exposure which rec-

ommend that they may be the most effective isolates for

sustainable insect control programs, (Yasin et al.,2017).

Endophytes have several advantages;  rstly, they are

inside the plant tissues and constantly shielded from

abiotic push factors. Also, the application cost is less

because of limited application through seed treatment

or seedling dip or foliar spray. Moreover, once settled

as an endophyte, they may offer season-long protection

against the pests that have secretive life cycle by causing

encouraging discouragement or antibiosis (Vega et al.,

2008) or prompting their mortality or less pervasion. All

these properties of fungal endophytes make them rea-

sonable to be utilized as a bio-control agent to protect

crops from the pests. Insect pests such as the European

corn borer (Ostrinia nubilalis) in U.S.A., and the banana

weevil (Cosmopolites sordidus) in Uganda respectively

were effectively controlled by endophytic establishment

of B. bassiana.

Date palms are considered as the image of life in the

leave, since it endures high temperatures, and saltiness

when contrasted with numerous other fruit crop species.

One of the most established relationships that man has

had with a tree has been with date palms, which have

been developed since ancient times (Zohary and Hopf,

2012). One of the character of date palm is that it can

adapt to extreme drought, to heat, and to relatively high

levels of soil salinity. Nevertheless, extreme quantities

of salinity due to irrigation with saline water result to

a signi cant decrease in the productivity of the fruits.

It is important to study the mechanism of tolerance to

these abiotic stress in order to develop future date palm

varieties that can tolerate excessive soil salinity (Yaish

& Kumar 2015 ).

Several insects invade date palm trees, of which the

red palm weevil (RPW), R. ferrugineus (Olivier) (Coleop-

tera: Curculionidae), is a standout amongst the most

essential and harming pests, being a noteworthy risk to

date palm trees everywhere throughout the world. The

red palm weevil is ordinarily well hidden, and numerous

local pervasions have just recently been perceive, mak-

ing the red palm weevil a pest of major economic sig-

ni cance in all Persian Gulf Countries. This study aimed

to isolation and screening of antagonistic B. bassiana

from diverse soils and date palm and plantation crop

ecosystem in Hail region, Saudi Arabia.

MATERIAL AND METHODS

Soil samples, samples of infected and dead red palm

weevil (RPW) insects and date plant materials collected

according to RPW incidence map of Hail, Saudi Arabia

during the period 2016-2017. The B. bassiana (BH-2)

strain was isolated from soil sample collected from Al

Koutha village in Hail region. About 500 grams of soil

sample were placed in a plastic bag. Five larvae (L3-L4)

of the red palm weevil R. ferrugineus were added to the

soil substrate to be infected by the entomopathogenic

fungi. The plastic bag was kept for 2-3 weeks at room

temperature (20-25°C) and a ca. 80% humidity level was

ensured inside the bag by periodic water sprays. Dead

larvae were collected, placed on wet  lter paper in Petri

dishes and put in an incubator at 25°C. The mycelium

and spores that emerged on larvae were transferred

directly onto growth medium (PDA – PotatoDextrose

Agar) in Petri dishes and incubated at 25°C for 10 days;

the isolates were then puri ed by repeated transplanting

(Goettel and Inglis, 1997).

The B. bassiana (BH-1, BH-2 and VBM) isolates were

obtained from naturally infected RPW adults collected

in from Jubbah village in Hail region (Fig 1). To promote

conidial growth, mycosed RPW adult cadavers were

placed separately on  lter paper soaked daily with water

to achieve ca.100% RH inside Petri dishes. The petri

dishes were incubated at room temperature (20-25C°).

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

BEAUVERIA BASSIANA

ISOLATES 395

Khalid A. Asiry et al.

Parts of fungal propagules grown on the cadavers were

then transferred, with sterile needles, into Petri dishes

with SDAY1/4 (Sabouraud Dextrose Agar Fluka supple-

mented with yeast extract ¼of concentration) and kept

in an incubator at 25°C. Pure fungal colonies were then

stored on PDA (Potato Dextrose Agar) and MEA (Malt

Extract Agar) slants in bacteriological glass tubes at 4°C.

The B. bassiana isolates were con rmed by sequencing

analyses of the 18SrRNA gene and the internal tran-

scribed spacer (ITS1).

For microscopic identi cation of the fungus, pure fun-

gal culture was used after successive puri cation. With

the help of inoculating needle some portion of growth

of the fungus was teased and place it on the slide then,

70% ethanol was used for washing, then ethanol was

removed by blotting paper. Then, a drop of Lacto-phenol

cotton blue was kept, and the mycelium was spread with

needles, and a cover slip placed and examined under

microscope with high power objective. The morphology

and spore structures were noted.

API 20 C AUX kit was used to identify the strains

of yeast isolated from RPW samples according to the

manufacturer’s instructions. Yeast strains were puri-

 ed by culturing them on PDA (Potato Dextrose Agar)

medium and incubated for 72 hours at 25°C. The strains

were puri ed twice before identi cation. After 48 and

72 hours the growth was compared in each cupule to

the 0 cupule, which is used as a negative control. A

cupule more turbid than the control indicates a posi-

tive reaction to be recorded in the result sheet. Each

strain was identi ed with the identi cation software by

manually entering the 7-digit numerical pro le via the

keyboard.

The molecular analyses for the fungal isolates were

performed at King Faisal University- the College of Agri-

culture and Food Science – Pest & Plant Disease Unit.

All isolates were re-grown on other PDA petri dishes

and inoculated on Potato dextrose broth medium for

microscopic examination (Fig 2), also for extraction of

DNA and molecular identi cation.

DNA wa s extracted using modi ed method of Del-

laporta and Hicks, 1983 as the following protocol:

Twenty mgs of frozen- dried mycelium or fresh har-

vested mycelium were ground with Kontes pestles in a

1.5 ml tube with 500 l of Dellaporta buffer (100 mMTris

pH 8. 50 mMethylenediamine-tetraacetate EDTA, 500

mMNaCl, 10 mM beta mercaptoethanol (BME). Thirty

three l of 20% sodium dodecyl sulfate (SDS, w/v) were

added, and incubate the mixture was vortexed and incu-

bated for 10 min at 65°C.160l of 5 M potassium acetate

KoAc (Sigma chemicals) were added and vortexed. The

mixture was spun for 10 min at 10,000 rpm in a micro-

centrifuge tube. 450 l of supernatant were transferred

to a new tube. 450 l phenol, chloroform and isoamyl-

alcohol (PCI) 25:24:1 were added and vortexed for 5min

and then spun for 5 min at 10,000 rpm. 400 l of the

upper phase were removed to a clean micro-centrifuge

tube and 0.5 volumes of isopropanol were added, vor-

texed and spinet for 10 min at 14,000 rpm. The superna-

tant was removed, the total nucleic acid was precipitated

in the bottom of the tube. The pellet was washed with

70% ethanol and spun 5 min at 10,000 rpm. The pellet

was resuspended in 100 l of ddH2O.

Two primer pairs, the forward IT5 primer 5’-GGAA-

GTAAAAGTCGTAACAAGG-3’) and the reverse ITS4

primer (5’-TCCTCCGCTTATTGATATGC-3’) were used to

FIGURE 1. Fungal growth of red palm weevil in Hail region.

Khalid A. Asiry et al.

396 ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

BEAUVERIA BASSIANA

ISOLATES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

amplify the entire ITS region (White et al., 1990). PCR

was done in a 25 µl reaction containing 1 µl of the fun-

gal DNA extract (40 ng of total DNA), 2 mM MgCl2, 2.5

of 10x PCR buffer,1.5 µL of 10 µM of each primer, 2.5 µl

of 10 mMdNTPs, 0.3 µl of 5U Taq DNA Polymerase and

the reaction was completed to 25L with Nuclease-free

water. PCR was conducted in the ESCO Swift Maxi Ther-

mal Cycler with initial denaturation at 95°C for 2 min,

followed by 35 cycles of 95°C for 30 sec, 52°C for 30 sec,

and 72°C for 30 sec, and the  nal cycle is a polymer iza-

tion cycle performed at 72°C for 10 min. PCR Products

were puri ed using QIAquick® PCR Puri cation Kit (Cat.

No. 28106) according to manufacturing procedures.

The puri ed PCR products were sequenced by Mac-

rogen Inc., (Korea), and sequencing of the puri ed iso-

lates was performed in both directions using ITS5 and

ITS4 primer pairs. Sequence alignments were edited by

MEGA6 (Tamura et al., 2013).

RESULTS AND DISCUSSION

Based on the API 20 AUX test performed on selected iso-

lates of Beauveria spp. (Table 1), Beauveria basiana was

the predominant yeast strain in all samples, also there

was small percentage of Aspergillus spp. On the basis

of the API 20 AUX test, the isolated yeast was identi ed

as Beauveria basiana. Its pro le makes up to 90% of

the strains. As shown in the table, various assimilation

pro les were obtained for Beauveria basiana. Studies on

assimilation pro les were based on the acidi cation of

FIGURE 2. Plates shown Beauveria bassianaafter re-grown in PDA in

PPDUlab (A,B). C and D B. bassianaas received form University of Hail.

Table 1. The biochemical pro le of Beauveria spp.

Sugar Reaction Sugar Reaction Sugar Reaction

esculin - Adonitol + salicin +

D-arabinose - rhamnose + glycogen -

dulcitol - Inositol + dextrose +/-

D-xylose - Lactose + trehalose +

raf nose - sorbitol + maltose +

galactose + dextrine +/- sucrose +

D-fructose + mannitol - dolicitol -

Note: + = Positive reaction

+/- = Weak reaction

- = Negative reaction

Khalid A. Asiry et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

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ISOLATES 397

twenty sugars. The results indicate that galactose, fruc-

tose, rhamnose, maltose, sucrose and trehalose were

assimilated at high degree contrasted with esculin, ara-

binose, xylose, raf nose, mannitol, glycogen and dulci-

tol. Biochemical properties and speci cally their use of

sugars can be utilized to help the morphology and are

valuable for recognizing species of Beauveria. Mugnai

et. al. (1989) contemplated the intra- and interspeci c

variety of 32 isolates appointed to the genus Beauveria.

They presumed that cultural characters were profoundly

factor also, couldn’t be utilized dependably to separate

species.

Depending on cultural and microscopic examination,

fungal isolates belonged to two genera Beauveria and

Aspergillus as shown on Table (2).

All extracted DNAs from the three fungal isolates

gave a clear bands on the expected size ≈ 600 bp

using primer pairs ITS5&ITS4 (Fig 3). Blast analysis

revealed that the fungal isolates are Beauveria bassiana.

Sequences alignment (Fig 3) showed that all isolates

had same sequence with 100% similarity with chinses

isolates (Accession No. JQ320361). All sequences of the

three isolates were deposited in the Genbank. 16 S rDNA

nucleotide sequence has been sent to the Genbank for

sequence publication.

The red palm weevil R. ferrugineus is a standout

amongst the most serious pests of various date palm

species, including date palms (Giblin-Davis, 2001). The

weevils create inside the tree trunk, wrecking its vascu-

lar system and in the long run causing the crumple and

death of the tree. The pest is generally circulated in Oce-

ania, Asia, Africa and Europe. The RPW makes extreme

harm to coconuts in Southeast Asia (Giblin-Davis, 2001).

It showed up in the Middle East in the 1980s and has

vigorously harmed date production by pulverizing huge

number of date palms (Murphy and Briscoe 1999). Inva-

sion was  rst reported in Israel and Jordan in 1999

(Khan and Gangapersad 2001).

The enormous economic losses created by RPW for

date palm trees in Hail region encouraged the authors

Table 2. Fungal speices isolated from R. ferrugineus

in Hail region.

No Label Colour Fungus name

1 BSA-1 white Beauveria bassiana

2 BH-1 brown Aspergillus ssp.

3 BH-2 white Beauveria bassiana

4 BH-3 white Beauveria bassiana

5 VBM green Aspergillus ssp.

FIGURE 3. PCR products of ITS region using ITS5 and ITS4 primer pairs. M: 100 bp

DNA Ladder; -Ve is the non-template control while +Ve is the positive control, BSA-

1, BH-2 and BH-3 are Beauveriabassiana isolates from Hail.

Khalid A. Asiry et al.

398 ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

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ISOLATES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

to seek for an economically feasible technique to be

employed for eradicating this harmful insect. During

surveying the various infected date palm farms, many

dead RPW were found under the infected trees covered

with whitish material. In addition, farmers informed the

researchers that they do not use any chemical treatment

for pest control in their farms. This information moti-

vated the researchers to try to recognize the causes of

RPW death. Hence, the research has been resumed by

collecting the dead insects are identifying the whitish

material covering their bodies. The morphological tests

indicated that the whitish materials were fungus growth.

Subsequently, the biochemical identi cation tests indi-

cated that the fungus was Beauveria bassiana. Then, the

identi cation was con rmed using molecular biology

tests.

Fungi are the commonest reason of insect disease

in nature. Certain species of entomopathogenic fungi

shown speci city to certain host (Wang, Chengshu &

Wang, Sibao. 2017). Fungi may infect insects by direct

in ltration of the cuticle thus works as contact insecti-

cides. Beauveria is standout amongst other known gen-

era of entomopathogenic fungi and worldwide various

enrolled mycoinsecticide formulations in view Beauveria

bassiana is utilized for control of insect pests (Thomas

and Read, 2007). This fungus has an especially wide host

range (over 700 species) enabling it to be utilized against

vectors of human disease and an extensive variety of

insect pests (de Faria, and Wraight, 2007). For instance

in China, around one million hectares a year are treated

with Beauveria bassiana to control forest insects such

as the pine caterpillar Dendrolimus punctatus (Wang

et al., 2004)

Beauveria is outstanding for creating huge cluster of

biological active secondary metabolites including non-

peptide pigments and polyketides (e.g., oosporein, bassia-

nin and tenellin), nonribosomally synthesized peptides

(e.g., beauvericin, bassianolides and beauveriolides), and

discharged metabolites associated with pathogenesis and

destructiveness (e.g., oxalic acid) that have potential or

acknowledged modern, pharmaceutical and agricultural

uses (Xu et al., 2009). The mechanism of action and bio-

logical function of Oosporein have persisted unclear.B.

bassianahas developed the ability to parasitize insects.

A unique zinc  nger transcription factor, BbSmr1 (B.

bassianasecondary metabolite regulator 1), was identi-

 ed in a screen for oosporein overproduction. Deletion

of Bbsmr1 resulted in up-regulation of the oosporein

biosynthetic gene cluster (OpS genes) and constitutive

oosporein production, (Fan et al., 2017).

The dominant part of endophyte explore has concen-

trated to date on the vertically-transmitted endophytes

inside the genus Neotyphodium (Clavicipitaceae) that

systemically colonize the over the ground parts of a few

grasses. These clavicipita-ceous endophytes are gener-

ally known to present a variety of potential advantages

to their grass host plants (Kuldau and Bacon, 2008). Less

consideration has been given to the evenly transmitted

non-clavicipitaceous endophytes, which are in nature

and commanded by the Ascomycetes (Arnold and Lut-

zoni, 2007); of which a few genera are fungal ento-

mopathogens (Ascomycota: Hypocreales).

Rising as an energizing

new area of research,

‘fungal ento-mopathogens as endophytes’ has

just

rather recently been incorporated into

a more than

100

old endophyte research base after the

recupera-

tion

different

genera of fungal ento-mopathogens

as endophytes from

various

plant species (Vega

et al., 2008). Some of these fungi have been

accounted

for

normally happening endophytes

, while others

have been

brought into the plant utilizing distinctive

different inoculation techniques. Spearheading

take a shot at entomopathogenic endophytes was

directed with B. bassiana (Balsamo), a universal

soil-borne fungus that infects an extensive variety

of various insects (>700 insect species; Inglis et al.,

2001) and is a standout amongst the most marketed

fungal biopesticides (de Faria and Wraight, 2007).

Lewis and Cossentine (1986) credited the season-

long concealment of the European corn borer Ostrinia

nubilalis (Hübner) (Lepidoptera: Pyralidae) in maize Zea

mays L. (Poaceae), estimated as reduced tunneling by

the insect, to the establishment of B. bassiana as an

endophyte following utilization of a watery suspen-

sion of the fungus to the plants. Ensuing work by Lewis

and colleagues utilizing a similar model system dem-

onstrated fruitful re-isolation of B. bassiana from inte-

rior plant tissues after utilization of the fungus utilizing

different inoculation methods. These antagonist fun

can have bene cial effects on host plants, e.g., plant

growth promotion, reducing disease severity, inducing

plant defense mechanisms, and producing anti-herbi-

vore products (Arnold and Lewis, 2005, Amatuzzi, et al.,

2017).

In addition to maize, a wide assortment of host plants

(counting both agronomic and weed species) have addi-

tionally been appeared to harbor B. bassiana as an endo-

phyte. As opposed to B. bassiana, the host plant scope of

other fungal entomopathogens is as yet developing. For

example, Verticillium (=Lecanicillium) lecanii (Zimm.)

Viegas has been accounted for as a natural endophyte

in bear-berry Arctostaphylos uva-ursi L. (Ericaceae)

(Widler and Muller, 1984) and ironwood (Bills and Pol-

ishook, 1991). A relatively recent development, the use

of some fungal entomopathogens could function as

biofertilizers. Various inoculation techniques (e.g., foliar

sprays, soil drenching, seed soaking, injections, etc.)

are effective in introducing fungal entomopathogens as

Khalid A. Asiry et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF INDIGENOUS

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ISOLATES 399

endophytes, but colonization appears to be localized and

ephemeral, (Vega 2018).

There is presently considerable con rmation that some

endophytic fungal entomopathogens, especially B. bassi-

ana and Lecanicillium spp. (some time ago Verticillium

lecanii), may likewise exhibit hostile action against plant

pathogens , in addition to their outstanding biocontrol

activity against insect pests. This proposes that these

entomopathogens have a promising potential to be devel-

oped as biopesticides for numerous reasons in IPM meth-

odologies (Goettel et al., 2008; Vega et al., 2009; Ownley

et al., 2010). Beauveria bassiana strain 11–98, applied as

a seed treatment, has been accounted to suppress damp-

ing-off caused by the soil-borne pathogens, Rhizoc-tonia

solani Kuhn (Basidiomycota: Cantharellales) and Pythium

myriotylum Drechsler (Oomycota: Pythiales), in tomato

(Ownley et al., 2004; Clark et al., 2006) and cotton seed-

lings (Grif n, 2007; Ownley et al., 2008). Pre-treatment of

cotton seedlings with the same B. bassiana strain likewise

resulted in reduced seriousness of bacterial blight caused

by Xanthomonas axonopodis pv. malvacearum (Xam)

(Grif n et al., 2006; Ownley et al., 2008).

More recently, several strains of B. bassiana were

found to fundamentally diminish the rate and serious-

ness of the Zucchini yellow mosaic virus (ZYMV; genus

Potyvirus, family Potyviridae) in squash (Jaber and

Salem, 2014) and downy mildew caused by Plasmopara

viticola (Berk. and Curt.) Berl. and de Toni. (Oomycota:

Peronosporaceae) in grapevines (Jaber, 2015) following

foliar inoculation of plants with conidial suspensions of

the tested strains.

CONCLUSION

Strains of Beauveria bassiana (Bals.-Criv.) Vuill. and

Metarhizium anisopliae (Metchn.) Sorokin (Hypocreales:

Clavicipitaceae) have been isolated from wild R. ferrug-

ineus populations (Lo Verde et al., 2015). Beaveria bassi-

ana fungus could be used as one of the biological strate-

gies in controlling red palm weevil (Hajjar et. al., 2014).

Based on the morphological, biochemical and molecu-

lar biological techniques, B. bassiana was isolated and

identi ed as the fungal strain for the  rst time in Hail

region, Saudi Arabia in which this fungal strain can be

utilized in the biological control of the red palm weevil

R. ferrugineus which is considered as a destructive pest

of date palms in this region.

ACKNOWLEDGEMENTS

The authors would like to express sincere thanks and

gratitude to the Al-Jomaiah’s Chair of Sustainable

Development in Agricultural Communities at the Uni-

versity of Hail for providing  nancial support.

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