Egypt. J. Plant Prot. Res. Inst. (2019), 2 (3): 450 - 459
Egyptian Journal of Plant Protection Research Institute www.ejppri.eg.net
Molecular identification of the entomopathogen Aspergillus candidus and its pathogenicity to the mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae) in Egyptian sugar beet fields
Bazazo, K.G.I.1; Amira, S.H.M. Ibrahim2 and Ismael, A. Khatab3 ¹Plant Protection Research Department, Sugar Crops Research Institute, Agricultural Research Center, Egypt. ²Economic Entomology Department, Faculty of Agriculture, Kafr El-Sheikh University, Egypt. ³Genetics Department, Faculty of Agriculture, Kafr El-Sheikh University, Egypt. ARTICLE INFO Abstract: Article History Several host plants are subject to infest with Received: 2 / 7 / 2019 Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), Accepted: 26 / 9 /2019 from which is sugar beet. The infested plants suffer from ______reduction in photosynthetic processes. Biological control is an Keywords essential approach to control. The fungus Aspergillus candidus Molecular identification, is one of the entomopathogens attacking this mealybug. The pathogenicity, Aspergillus, current investigation was done at "Kafr Ascar" village, Kafr rDNA and Phenacocus El-Sheikh Governorate, Egypt during 2016/2017 and solenopsis. 2017/2018 seasons. Obtained results indicated that the natural mortality of P. solenopsis adults due to the fungus A. candidus infection in the field ranged between 2-19 individuals/ 50 plants from 25 October to 10 May, 2016/2017 season and the total number of dead insects recorded throughout the season was 71 individuals/ 300 plants. According to statistical analysis, the results indicated that the temperature and humidity had a significant effect on the activity of the fungus and therefore on mortality rates.The entomopathogenic fungus was isolated and identified by GATC (Biotech Sequence Company, Germany), as A. candidus strain HNMF075 (Ascomycetes: Eurotiaceae) for the first time in Egypt. Suspension of this fungus was made, adjusted to 5 spores/ ml water, for using in laboratory and field tests.
In a laboratory tests, the mortalities of adults were 55.00 and
95.00 two and four days after treatment, respectively. The
mean of mortality was 75.00 %. Also, in a field tests, the
reductions of adults were 53.00 and 67.42% two and four days
after treatment, respectively, with 60.21% average reduction
based on DNA sequences using rDNA, data showed that the
Aspergillus isolate belonged to A. candidus with a high
similarity of 94%. It is concluded that A. candidus strain
HNMF075 is an effective biological agent against P. solenops
is in sugar beet fields in Egypt.
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Introduction The cotton mealybug Phenacoccus over other insect pathogens like bacteria solenopsis Tinsley (Hemiptera: and viruses. Mass production techniques Pseudococcidae) is a polyphagous insect of fungi are much simpler, easier and feeding on more than 202 host plant cheaper than those of bacteria and virus. species from 55 families (Fand and Fungi unlike bacteria or viruses directly Suroshe, 2015). Large populations of infect through insect cuticle and do not mealybug cause general weakening, require ingestion for infection and also defoliation and death of susceptible sucking insects are infected (Pucheta et plants. As well, the honeydew excreted al., 2016). Entomopathogenic fungi play by the mealybugs caused growth of sooty an important role in the natural pest moulds and other secondary infections control in various crops through that reduce photosynthesis and impair epizootics; more than 750 species are the marketability of plant products pathogenic against insects (EL-Husseini (Ibrahim et al., 2015). P. solenopsis has et al., 2003 and 2004 and Ramanujam et a wide geographical distribution, Central al., 2014). Aspergillus candidus Link as America, the Caribbean, Ecuador, Chile, entomopathogen against different insect Argentina and Brazil (Culik and Gullan pests such as, Thaumetopoea wilkinsoni 2005). In Egypt, the occurrence of P. Tams (Lepidoptera: Notodontidae) solenopsis infestation was recorded on (Kenneth and Olmert, 1975); Coccus weed plants for the first time byAbd- hesperidum L. (Hemiptera: Coccidae) Rabou et al. (2010). In sugar beet (Beta (Samsinakova and Kalalova, 1975); vulgaris L.) fields, Vennila et al. (2014) Indarbela spp. (Lepidoptera: showed that pigweed Chenopodium Metarbelidae) (Singh and Singh, 1982); albam L. and spinach Spinacia oleracea Carvalhoia arecae Miler (Heteropetera: L. are host plants of P. solenopsis. Miriidae) (Dhileepan et al., 1990). Anonymous (2009) recorded that Finally, research on microbial pathogens mealybug is one of the insect pests of of insects is increasing considerably in sugar beet plants in Egypt. Also, Bazazo recent times to find out environmental et al. (2017) identified the specimens of friendly alternatives to hazardous P. solenopsis at Plant Protection chemical insecticides (Ramanujam et al., Research Institute, Agricutural Research 2014). Center. They estimated the numbers of Aspergillus spp. is commonly P. solenopsis infested leaves as 15-18 worldwide and includes industrially and symptoms/200 sugar beet plants at some biological control important members. It fields at Kafr El-Sheikh Governorate. is very important to isolate and identify Mealybugs are difficult to control with microorganisms to be used as biological insecticides, as they are able to rapidly control agents against insect pests. develop resistance and exhibit cryptic Moreover, morphological identification behavior. Further, they are covered by of microbial isolates could be waxes that protect their bodies from insufficient for distinguishing the insecticide penetration (Franco et al., species. PCR and DNA sequences are 2004). The low efficacy of insectcide use speed, sensitive and specific way for has been encouraged biocontrol identification and are proved extremely strategies (Daane et al., 2004). The useful in assessing the changes in fungus application of entomopathogenic fungus such as Aspergillus and can also provide was a valuable method of biological useful taxonomic information (Rasime, control of mealybugs (Mustu et al., 2016). Abundant gene regions, such as 2015). Fungal pathogens have certain rDNA regions could be used for advantages in pest control programms sequencing and molecular identification
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(Munusamy et al., 2010; Peterson, 2012 The daily records of temperature and Schoch et al., 2012) and there are (˚C), relative humidity (RH%) and rain precise analyses for rDNA for fall (mm/day) during the experimental differentiation of Aspergillus (Sabreen et season were obtained from al., 2015). Moreover, internal transcribed Meteorological Department at Sakha, spacer (ITS), as called, rDNA is Agricultural Research Station. Monthly commonly used for taxonomic studies, means of these factors were calculated. barcode gene for fungal identification The relationship between these weather and phylogenetic studies (Krijgsheld et factors and the number of dead mealybug al., 2013 and Rasime, 2016). adults was statistically calculated. Linear This study was carried out to correlations were fitted using IBM SPSS molecularly identify Aspergillus from Statistics 19 software to reveal the dead P. solenopsis picked up from the correlation between mortality rates of P. suger beet fields. Moreover, the solenopsis adults at respective weather virulence of Aspergillus against the factors. insect pest was assessed under 3. Isolation of entomopathogenic laboratory and field conditions. fungus: Materials and methods The dead insects were sterilized 1.Recording the dead Phenacoccus using 0.5% sodium hypochlorite and solenopsis adults with Aspergillus 75% ethyl alcohol and then rinsed in candidus: plenty of distilled water and dried with The dead individuals of P. solenopsis sterile filter paper. Then, the dead insects were recorded at the experimental field were individually kept in petri dishes (9 (about two feddans). Sugar beet, sultan cm diameter) provided with moistened cultivar sown on September, 2016 filter paper and incubated at 28ºC for at Kafr Ascar village, Kafr El-Sheikh three days to stimulate the growth of Governorate.They were picked up by a fungus.The pathogen was introduced into fine brush and preserved in small a petri dishes (9 cm diameter) having sterilized vials, as monthly samples water agar medium and incubated for beginning from October to three days under 28 ºC. Finally , a piece of agar with mycelial growth was April 2017, for 50 plants per inoculated using a sterilized needle into inspection. petri dishes having Potato Dextrose Agar 2.The effect of certain climatic factors (PAD) medium, according to Dourou- on Aspergillus candidus activity: Kpindou et al. (1995) (Figure,1).
Figure (1): Phenacoccus solenopsis adults with fungal hyphae of Aspergillus candidus strain HNMF075.
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4. Spore isolation for using in min, 35 cycles of 95 °C for 1.30 min, laboratory and field tests: 55°C for 45 sec, 72°C for 1 min. then The petri dishes having sporulated 72°C for 7 min. PCR products were fungi were washed with distilled water to separated by 1.2% agarose gels exclude the fungus and formulate as a electrophoresis, which run with 1X TAE spore suspension that was adjusted buffer. The 1Kbp DNA ladder was also to spores/ml water, using the run on each gel as a molecular weight micrometer slide, to be used in tests standard. The amplification of 5.8S against P. solenopsis adults. ribosomal RNA gene and internal 5. Laboratory tests: transcribed spacer was done from fungal Forty P. solenopsis adults genomic DNA by PCR. PCR purified (20 petri dishes) for each sampling products of the rDNA of the strains were date, containing a piece of sugar beet leaf analyzed for nucleotide sequence were treated with A. candidus spores determination by using ABI PRISM® suspension by hand sprayer (1 liter). The 3500XL DNA Sequencer (Applied mortalities (%) were recorded two and Biosystems). four days after treatments according to 8. Data analysis and phylogenetic Samsinakova and Kalalove (1975). The analysis: fungus was reisolated from the dead A pairwise comparison among mealybug adults. isolate and other sequences from 6. Field tests: GenBank database with the BlastN The experimental area algorithm to determine relative phylogenetic positions was performed to (approximately 200 ) of sugar beet produce a dendrogram using neighbor- was divided into two plots. Each plot joining (NJ) trees. was divided into four replicates. One plot Results and Discussion was sprayed with A. candidus spores 1. Recording the natural mortalities of suspension by hand sprayer (1 liter). The Phenacoccus solenopsis adults with other one was left untreated (as a Aspergillus candidus: check).The number of dead mealybug Data in Table (1) showed that the individuals were recorded two and four number of dead adults was low on days after treatment according to October 25th and November 16th (3 and 6 Samsinakova and Kalalova (1975). The th individuals/50 plants, respectively). The spraying was done on 10 January 2018. number jumped to 15 individuals/50 The reductions were calculated using plants on December 18th. The dead Henderson and Tilton (1955) formula: numbers reached 17 and 19 individuals/50 plants on January 19th and February 20th, respectively. The 7. DNA isolation, PCR and sequencing: numbers decreased sharply and reached Total DNA was isolated using CTAB to 9 and 2 individuals/50 plant on March method Doyle and Doyle (1990). 23rd and April 10th, Concentration of obtained DNA (ng/μl) respectively.Throughout the season, the and purity were determined on total number of dead adults was 71 Nanodrop-photometer, and then kept at o individuals/ 300 sugar beet plants. 20 C till use. Polymerase Chain Reaction 2. The effect of certain weather factors (PCR) was done in a volume of 50μl on Aspergillus candidus activity: using 60ng genomic DNA, 0.2 A fitted linear regression model mMdNTP, 1.5 mM MgCl2, 5 pmol of showed that the mortality rates of P. primer and 0.5U Taq polymerase. PCR solenopsis adults were positively conditions were as follows: 95°C for 5 correlated with the temperature (y = 0.7
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Egypt. J. Plant Prot. Res. Inst. (2019), 2 (3): 450 – 459 x + 16, r2 = 0.825, p < 0.001) and (y = enhanced the role of the 0.6 x + 80, r2 = 0.516, p < 0.001). These entomopathogenic fugus in killing P. results suggested that the high solenopsis adults. temperature and relative humidity Table (1): Natural mortality of Phenacoccus solenopsis adults collected from 50 sugar beet plants /sampling date due to Aspergillus candidus during 2016/2017 season. No. of Phenacoccus Temperature Humidity Rain Date Mortality% solenopsis (oc) (RH %) (mm/day) dead October, 25 3 4.23 29.44 80.71 - November,16 6 8.45 25.82 91.28 - December,18 15 21.13 20.07 90.14 7.1 January, 19 17 24.94 18.82 91.71 8.1 Feburay, 20 19 26.76 17.01 90.00 8.7 March, 23 9 12.68 20.44 91.00 - April, 10 2 2.82 25.08 81.00 - Total 71 - - - - Pell et al., (2001) indicated that the Table (2) showed that the mortalities environmental conditions particularly, of adults were 55.00 and 95.00% two humidity and temperature play an and four days after treatment, important role in the infection and respectively. The overall mortality sporulation of entomopathogenic fungus. recorded throughout the experimental Humidity (RH %) is required for spore period was 75.00%. Roberts and Yendel germination and sporulation outside the (1971) and Pierre (1985) indicated that host. Most of the entomopathogenic Aspergillus spp. are imperfect fungi and fungi in tropical and subtropical areas are most frequently associated with require an optimum temperature of 25-30 insect diseases. Singh and Singh (1982) for successful control of insect pests. reported that A.candidus induced about Samsinakova and Kalalova (1975) 100% mortality under laboratory reported that the relatively high natural conditions against Indarela spp. Insects. mortality of scale insects depends on Also, Dhileepan et al. (1990) showed temperature and humidity conditions. that A. candidus is a virulent Also, Dhileepan et al. (1990) showed entomopathogen to C. arecae killing that the possible reason for the wide 50% within two days of inoculation and spread incidence of A. candidus against 100% in four days after inoculation. the spindle bug, Carvalhoia arecae in Finally, Samsinakova and Kalalova the field during the raining season. (1975) proved that A. candidus caused 3. Pathogenicity of Aspergillus about 85% to 100% mortality against candidus to Phenacoccus solenopsis in Coccus hesperidum L. (Hemiptera: laboratory tests: Coccidae) insect, two days after treatment under laboratory conditions.
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Bazazo et al., 2019 Table (2): Mortality percentages of Phenacoccus solenopsis adult treated with Aspergillus candidus suspension ( ) /ml water in the laboratory, 2017/2018. Duration after No.of adults before No. of dead adults Mortality treatment (day) treatment after treatment (%) 2 20 11 55.00 4 20 19 95.00 Overall 40 30 75.00
4.Effect of Aspergillus candidus fungi were effective microbial control suspension on Phenacoccus agents in crops with vegetation solenopsisin the field tests: contributes to the presence of high Table (3) indicate that the reduction relative humidity in the microclimate of P. solenopsis opulations were 53.00 within plants as occurring in sugar beet and 67.42% two and four days after fields. Ramanujam et al. (2014) showed treatment, respectively. Overall mean of that entomopathogenic fungi play an reduction was 60.21%. El-Husseini important role in the natural pest control (1981) proved that the entomopathogen in various crops through epizootics. Table (3): Reduction of Phenacoccus solenopsis due to Aspergillus candidus suspension ( ) /ml water in the field, 2017/2018 season. Treatment Aspergillus candidus Control Before spray Total 17 16 Mean 4.25 4.00 Total 11 22 After 2days Mean 2.75 5.50 Red.(%) 53.00 - Total 9 26 After 4days Mean 2.25 6.50 Red.(%) 67.42 - Overall mean of reductions 60.21 - 5. Molecular identificaion: other sequences in GeneBank. Based on The method to identify isolated BLST, the selected isolate was closely fungus at the species level was related to various species of Aspergillus developed using the rRNA genes. The and has 94% identical with A. candidus partial sequence with 600bp was (Figures , 2 and 3). amplified, sequenced and compared with
Figure (2): Phylogenetic tree of the nucleotide sequences of the PCR product of rRNA gene from BLAST.
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Moreover, Aspergillus sp. taxonomy is (Hunt et al., 2004). Sequences of rDNA complex, which makes its identification regions and phylogenetic analysis have unreliable as a result of intraspecific been used as targets for microbial isolates similarities (Gontia-Mishra et al., 2013). identification. It could be concluded that Therefore, molecular characterization the use of DNA sequences gives a better looks to be a confident tool in the picture of Aspergillus identification to be identification isolated fungus based on used as a biological control for insects. the sequencing of the 18 S rRNA genes
Figure (3): Aspergillus candidus isolate HNMF075 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence
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