Abstract

Beauveria spp. (one Beauveria sp., two B. brongniartii, and 47 B. bassiana) isolated from insects and soil from Kenya and other 16 different countries, were obtained from the CABI Bioscience Genetic Resource Collection. DNA was extracted from the 50 isolates and their genetic variability was investigated using restriction analysis of the internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP), ITS sequencing, and amplified fragment length polymorphism (AFLP). The B. bassiana isolates could not be distinguished by ITS-RFLP as all of them showed the same banding pattern. However, the AFLP technique provided more information on polymorphism between the isolates, allowing them to be clustered by relative similarity using band matching and unweighted pair group method with arithmetic mean analysis. Although no significant correlation between the isolates and host and geographical origins were observed, the technique revealed clonal populations of B. bassiana within Kenya.

Introduction

Fungal pathogens are capable of causing high levels of mortality in insect populations. As early as about 1000 AD, sericulturists in Asia reported Beauveria bassiana infections in silkworm [1].

B. bassiana is a naturally occurring mitosporic fungus with a wide arthropod host range. Many isolates have been studied because of their potential use as biopesticides [2–5] and B. bassiana has been adopted successfully to control a number of important pests. It controls insects by causing disease; spores of the fungus land upon the cuticle of susceptible insects, germinate and force a germ-tube through the cuticle by a combination of enzymatic action and physical pressure. Growth of the fungus inside the insect causes death by attrition and by disruption of physiological processes of the insect [6].

B. bassiana conidia have been isolated from soil at the base, and from bark of Elm trees that died from Dutch Elm Disease. It was suggested that these conidia had originated from insects, predominantly under the bark, that had died from B. bassiana infections. This soil survey for fungi suggests that neither B. bassiana nor Metarhizium anisopliae are frequently found except in locations that are known to harbour insect populations [7].

Beauveria comprises two main insect pathogenic species, B. bassiana and B. brongniartii, which are mainly parasitic on Lepidoptera and Coleoptera. Beauveria species are classified by the shape of their conidia and the placement of conidia on the conidiogenous apparatus [8]. Recent studies have distinguished at least six species of Beauveria using morphological and physiological tests [9], and natural variation in populations has been derived from esterase and acid phosphatase profiles which indicate some homogeneity among isolates from related hosts and from the same geographical area [10,11].

A recent study which compared electrophoretic profiles of extracellular iso-enzymes (enzymes involved in the degradation of complex material) with PCR banding patterns based on variable number tandem repeats revealed little genetic variability among B. bassiana isolates from the coffee berry borer Hypothenemus hampei, and this polyphasic analysis did not provide any simple correlation with host, geographic origin or pathogenicity [12].

A research based on RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) analyses showed clearer relationships between the population structure of B. bassiana and some defined host species [13]. However, a more recent research also based on RAPD analysis showed certain correlation between B. bassiana isolates and their geographical origin, but no clear correlation between those clusters and the insect host or pathogenicity against H. hampei[14].

The internal-transcribed-spacer ribosomal region (ITS)-RFLP technique has led to separation of B. brongniartii populations from diverse geographical and biological origins in previous studies [15]. Other molecular methods such as RAPD-PCR [16] have been used to characterise B. brongniartii.

However AFLP (amplified fragment length polymorphism) might be a more reliable molecular marker technique for specific identification of B. bassiana as it encompasses the analysis of the total genome, and the amount of information obtained is extensive. The AFLP technique on agarose-synergels has been used successfully to examine the interspecific variation within the invertebrate mycopathogen Nomuraea rileyi[17,18].

In this study, we used various B. bassiana isolates from various sources (insects and soil) from several different countries, which had been deposited in the CABI Bioscience Genetic Resource Collection. The purpose of this study was to investigate the genetic variability of the various B. bassiana isolates using an adapted AFLP technique on denaturing polyacrylamide gels in comparison with ITS-RFLP and ITS sequencing, as an attempt to both reveal molecular variation and clarify any correlation with host and geographic origin.

Materials and methods

Beauveria spp. isolates and growth conditions

The 50 Beauveria spp. isolates (one Beauveria sp., two B. brongniartii, and 47 B. bassiana) used are described in Table 1. All isolates are held in the CABI Bioscience Genetic Resource Collection of fungal strains both as freeze-dried and liquid-nitrogen stocks. These stocks were re-activated by addition of sterile water and culturing on Sabouraud dextrose agar (SDA) (Oxoid) or potato carrot agar (PCA) (20 g grated potato, 20 g grated carrot, 20 g agar Oxoid no.3, 1 l tap water) plates and incubated at 25°C.

1

Beauveria spp. isolates from CABI Bioscience Genetic Resource Collection used in this study

Isolate IMI number Geographical origin Host 
B. bassiana 348083 Portugal Phoracantha semipunctata 
B. bassiana 339137 Colombia H. hampei 
B. bassiana 382806 Kenya (Kiboko) soild 
B. bassiana 382723 Kenya (Gucha) S. zeamais 
B. bassiana 339140 Colombia H. hampei 
B. bassiana 372444 Uganda Cosmopolites sordidus 
B. bassiana 351836 Morocco Sitona discoideus 
B. bassiana 382471 Kenya (Bondo) S. zeamais 
B. bassiana 382807 Kenya (Kibwezi) soild1d 
B. bassiana 339138 Israel Phlebotomus papatasi 
B. bassiana 382294 Kenya (Nyamira) Tribolium sp. 
B. bassiana 341545 Brazil Anthonomus vestitus 
B. bassiana 382808 Kenya (Kiboko) Prostephanus truncatuse 
B. bassiana 382629 Kenya (Kuria) S. zeamais 
B. bassiana 382763 Kenya (Bondo) Tribolium sp. (passage) 
B. bassiana 382805 Kenya (Machakos) soil 
B. bassiana 361056 Costa Rica Coleoptera sp. 
B. bassiana 382809 Kenya (Voi) soil 
B. bassiana 382628 Kenya (Bondo) Tribolium sp. (passage) 
B. bassiana 382765 Kenya (Kisii) S. zeamais 
B. bassiana 382764 Kenya (Taita Taveta) S. zeamais 
B. bassiana 356817 India Coleoptera sp. 
B. bassiana 382626 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 382296 Kenya (Taita Taveta) S. zeamais 
B. bassiana 382627 Kenya (Nyamira) S. zeamais 
B. bassiana 382470 Kenya (Nakuru) S. zeamais 
B. bassiana 382295 Kenya (Trans-Nzoia) S. zeamais 
B. bassiana 382810 Kenya (Makindu) soil 
B. bassiana 382630 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 358840 England Otiorrhynchus sulcatus 
B. bassiana 327908 Kenya H. hampei 
Beauveria sp. 331275 USA (Montana) Grasshopper 
B. bassiana 160567 Australia Eucalyptus pauciflora 
B. brongniartii 334663 Kenya weevil 
B. bassiana 129066 Papua New Guinea Pantorhytes szent-ivanys 
B. brongniartii 228343 Malawi Phoracantha semipunctata 
B. bassiana 386705 Brazil (Bahia) Coleoptera sp. 
B. bassiana 386706 Brazil (Mato Grosso) Rommatocerus schistocercoides 
B. bassiana 386693 Philippines Leptocoris oratorius 
B. bassiana 386695 Ethiopia Schistocerca gregaria 
B. bassiana 386696 Brazil (Amazonas) Diabrotica sp. 
B. bassiana 386694 Portugal Popillia japonica 
B. bassiana 386699 Zimbabwe Neochetina biuchi 
B. bassiana 382724 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 382302 Kenya (Kisii) S. zeamais 
B. bassiana 386700 Kenya (Kibwezi) Prostephanus truncatuse 
B. bassiana 386701 Kenya (Kibwezi) S. zeamaisg 
B. bassiana 386704 Kenya (Kiboko) Bostrychid sp. 
B. bassiana 386703 Kenya (Kuria) S. zeamais 
B. bassiana 386702 Kenya (Uasin Gishu) S. zeamais 
Isolate IMI number Geographical origin Host 
B. bassiana 348083 Portugal Phoracantha semipunctata 
B. bassiana 339137 Colombia H. hampei 
B. bassiana 382806 Kenya (Kiboko) soild 
B. bassiana 382723 Kenya (Gucha) S. zeamais 
B. bassiana 339140 Colombia H. hampei 
B. bassiana 372444 Uganda Cosmopolites sordidus 
B. bassiana 351836 Morocco Sitona discoideus 
B. bassiana 382471 Kenya (Bondo) S. zeamais 
B. bassiana 382807 Kenya (Kibwezi) soild1d 
B. bassiana 339138 Israel Phlebotomus papatasi 
B. bassiana 382294 Kenya (Nyamira) Tribolium sp. 
B. bassiana 341545 Brazil Anthonomus vestitus 
B. bassiana 382808 Kenya (Kiboko) Prostephanus truncatuse 
B. bassiana 382629 Kenya (Kuria) S. zeamais 
B. bassiana 382763 Kenya (Bondo) Tribolium sp. (passage) 
B. bassiana 382805 Kenya (Machakos) soil 
B. bassiana 361056 Costa Rica Coleoptera sp. 
B. bassiana 382809 Kenya (Voi) soil 
B. bassiana 382628 Kenya (Bondo) Tribolium sp. (passage) 
B. bassiana 382765 Kenya (Kisii) S. zeamais 
B. bassiana 382764 Kenya (Taita Taveta) S. zeamais 
B. bassiana 356817 India Coleoptera sp. 
B. bassiana 382626 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 382296 Kenya (Taita Taveta) S. zeamais 
B. bassiana 382627 Kenya (Nyamira) S. zeamais 
B. bassiana 382470 Kenya (Nakuru) S. zeamais 
B. bassiana 382295 Kenya (Trans-Nzoia) S. zeamais 
B. bassiana 382810 Kenya (Makindu) soil 
B. bassiana 382630 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 358840 England Otiorrhynchus sulcatus 
B. bassiana 327908 Kenya H. hampei 
Beauveria sp. 331275 USA (Montana) Grasshopper 
B. bassiana 160567 Australia Eucalyptus pauciflora 
B. brongniartii 334663 Kenya weevil 
B. bassiana 129066 Papua New Guinea Pantorhytes szent-ivanys 
B. brongniartii 228343 Malawi Phoracantha semipunctata 
B. bassiana 386705 Brazil (Bahia) Coleoptera sp. 
B. bassiana 386706 Brazil (Mato Grosso) Rommatocerus schistocercoides 
B. bassiana 386693 Philippines Leptocoris oratorius 
B. bassiana 386695 Ethiopia Schistocerca gregaria 
B. bassiana 386696 Brazil (Amazonas) Diabrotica sp. 
B. bassiana 386694 Portugal Popillia japonica 
B. bassiana 386699 Zimbabwe Neochetina biuchi 
B. bassiana 382724 Kenya (Uasin Gishu) S. zeamais 
B. bassiana 382302 Kenya (Kisii) S. zeamais 
B. bassiana 386700 Kenya (Kibwezi) Prostephanus truncatuse 
B. bassiana 386701 Kenya (Kibwezi) S. zeamaisg 
B. bassiana 386704 Kenya (Kiboko) Bostrychid sp. 
B. bassiana 386703 Kenya (Kuria) S. zeamais 
B. bassiana 386702 Kenya (Uasin Gishu) S. zeamais 

Genetic Resource Collection, CABI Bioscience, Egham, UK.

Isolates were originally recovered from insect cadavers unless otherwise stated or referred to by (d, e, f and g). Both IMI 339137 and IMI 339140 isolates were recovered from the coffee berry borer H. hampei, and this host was referred as coffee berry on Fig. 2A.

Indicates the various districts in Kenya from where B. bassiana isolates were recovered [19]. IMI 386700 and IMI 386701 were respectively referred as KIB West 1P and KIB West 1S on both Fig. 2A and B.

Isolated from Galleria larvae through soil baiting.

Re-isolated from Prostephanus truncatus through pathogenicity test.

Originally isolated from S. zeamais and re-isolated (passage) through Tribolium sp.

Re-isolated from S. zeamais through pathogenicity test.

Sporulated cultures for regular use were kept on SDA plates at 4°C. Spore suspensions of young cultures were used for DNA extraction. The spores were resuspended in 0.05% Tween 80 (BDH) up to 107–108 spores ml−1 and 20 µl from this suspension were inoculated onto PCA or malt extract agar (20 g toffee barley malt extract, 20 g agar Oxoid no.3, 1 l tap water, pH 6.5) 5-cm plates. The mycelium was harvested after incubation for 2–3 days at 25°C and used for DNA extraction.

DNA extraction

Genomic DNA from each of the 50 Beauveria sp. isolates was extracted using the QIAmp® DNA Mini Kit (Qiagen, Germany). The mycelium from half a plate was resuspended in 600 µl TE buffer, homogenised using conical grinders and treated with 400 U of lyticase for 2 h at 30°C. After an overnight incubation with protease the protocol for one column extraction was followed according to the manufacturer instructions. The DNA was eluted in 100 µl of sterile water. The final DNA concentration was in the range of 20–200 ng µl−1 measured using the spectrophotometer GeneQuant pro RNA/DNA Calculator (Amersham Pharmacia Biotech UK Ltd.).

ITS-RFLP and sequencing

The ribosomal ITS regions ITS1 and ITS2 flanking the 5.8S subunit were amplified with the following pair of primers: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [20] or ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) [21] and ITS4 (the primers were synthesised by Amersham Pharmacia Biotech UK Ltd.). The reaction mixture consisted of 1 µl of B. bassiana genomic DNA, 50 pmol of each primer, a final concentration of 200 µM for each dNTP, 2.5 U of Taq polymerase (Sigma, St. Louis, MO, USA), 5 µl of 10× Taq Polymerase buffer, 2.5 mM MgCl2, and sterile Millipure water to 50 µl. The thermal programme comprised 1 initial cycle of denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 45 s and extension at 72°C for 2 min. An additional cycle comprising a 10-min extension at 72°C was included as a final step. The cycles were carried out in a HYBAID PCR Express thermal cycler.

Three µg of the PCR-amplified ITS regions were digested with each of the following restriction endonuclease enzymes HaeIII, HpaII, EcoRI, PstI, AluI and MboI (5 U per µg DNA) for 4 h at 37°C in the buffer recommended by the manufacturer (Promega, Madison, WI, USA). Digests were electrophoresed at 100 V for 3 h on a 3% MetaPhor agarose (BioWhittaker Molecular Applications, Walkersville, MD, USA), using Tris/acetate buffer (pH 8). A 100 bp DNA ladder (Gibco BRL, Grand Island, NY, USA) was used as size markers. After electrophoresis, the gels were stained in ethidium bromide (5 µg ml−1) for 15–30 min and photographed under UV light.

The ITS fragments for cloning and automated ABI sequencing were purified using Promega Wizard PCR Preps DNA Purification System. Full sequencing of the ITS regions was carried out for the 27 chosen isolates, and a neighbour-joining dendrogram was generated using Clustal W [22,23].

AFLP

An adapted version of the original technique [24] and application for fingerprinting of B. bassiana DNA is described in this study. All the adapters and primers used are described in Table 2, and they were purchased from Amersham Pharmacia Biotech, UK Ltd., Piscataway, NY, USA.

2

AFLP adapters, recombined adapters and primers used in this study

Name Sequence 5′–3′ Application 
EcoRI-ad1 CTCGTAGACTGCGTACC (forward) PCR pre-amplification 
EcoRI-ad2 AATTGGTACGCAGTCTAC  
EcoRI-adapters CTCGTAGACTGCGTACC Recombined adapters for ligation 
 CATCTGACGCATGGTTAA  
HpaII-ad1 GACGATGAGTCCTGAG (reverse) PCR pre-amplification 
HpaII-ad2 CGCTCAGGACTCATCGT  
HpaII-adapters GACGATGAGTCCTGAG Recombined adapters for ligation 
 TGCTACTCAGGACTCGC  
EcoRI-GC GACTGCGTACCAATTCGC (forward) PCR selective-amplification 
EcoRI-AT GACTGCGTACCAATTCAT (forward) PCR selective-amplification 
HpaII-CTC GATGAGTCCTGAGCGGCTC (reverse) PCR selective-amplification 
HpaII-ACC GATGAGTCCTGAGCGGACC (reverse) PCR selective-amplification 
Name Sequence 5′–3′ Application 
EcoRI-ad1 CTCGTAGACTGCGTACC (forward) PCR pre-amplification 
EcoRI-ad2 AATTGGTACGCAGTCTAC  
EcoRI-adapters CTCGTAGACTGCGTACC Recombined adapters for ligation 
 CATCTGACGCATGGTTAA  
HpaII-ad1 GACGATGAGTCCTGAG (reverse) PCR pre-amplification 
HpaII-ad2 CGCTCAGGACTCATCGT  
HpaII-adapters GACGATGAGTCCTGAG Recombined adapters for ligation 
 TGCTACTCAGGACTCGC  
EcoRI-GC GACTGCGTACCAATTCGC (forward) PCR selective-amplification 
EcoRI-AT GACTGCGTACCAATTCAT (forward) PCR selective-amplification 
HpaII-CTC GATGAGTCCTGAGCGGCTC (reverse) PCR selective-amplification 
HpaII-ACC GATGAGTCCTGAGCGGACC (reverse) PCR selective-amplification 

Five hundred ng of B. bassiana genomic DNA was incubated for 1 h at 37°C with 5 U EcoRI (rare-cutter) and 5 U HpaII (regular-cutter), 4 µl 10× Multi-core buffer (the restriction enzymes and buffer were purchased from Promega), add distilled sterile water up to 40 µl. Next, 10 µl of a solution containing 5 pmol EcoRI-adapters, 50 pmol HpaII-adapters, 1 U T4 DNA-ligase (Promega), 1 mM ATP, 1 µl 10× Multi-core buffer, and distilled sterile water up to 10 µl. The incubation was continued for 14 h at 37°C, after ligation the reaction mixture was stored at −20°C.

The reaction mixture for the pre-amplification consisted of 1 µl of the ligated B. bassiana DNA, 50 pmol EcoRI-ad1, 50 pmol HpaII-ad1, 500 µM for each dNTP, 1 U of Taq polymerase (Sigma), 2 µl of 10× Taq Polymerase buffer, 2.5 mM MgCl2, and sterile Millipure water to 20 µl. The thermal programme comprised one initial cycle of denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 1 min and extension at 72°C for 1.5 min. An additional cycle comprising a 5-min extension at 72°C was included as a final step. The selective amplification followed the original technique [24]. All amplifications were carried out in a HYBAID PCR Express thermal cycler.

Ten µl of microSTOP-red buffer (Microzone Ltd.) was added to the 20 µl selective-PCR product, heated at 95°C for 3 min, and 0.8 µl were loaded onto a Long Ranger 25 cm (0.25 mm) polyacrylamide gel and the bands were visualised on a 4200 LI-COR DNA Analyzer System. The 50–350 bp IRD700 Sizing Standard (LI-COR) was used as size markers. The AFLP banding patterns were analysed, and dendrograms showing the percentage of relative similarity between isolates were generated using the GelCompar II software (Applied Maths, Kortrijk, Belgium).

Results and discussion

ITS-RFLP and ITS sequencing

The B. bassiana isolates could not be distinguished using the ITS-RFLP technique as all of them showed the same banding pattern either using HaeIII, HpaII, EcoRI or PstI. Twenty seven isolates were chosen and digested with AluI or MobI and again the banding patterns were very similar. Only B. bassiana IMI 361056 showed distinct restriction enzymes sites and banding pattern for AluI and MobI. B. brongniartii IMI 334663 and IMI 228343 showed different patterns from B. bassiana, as expected. Table 3 shows all the sizes of the restriction digestion fragments. These results confirm that ITS regions have limited value as markers within B. bassiana since there is not enough variation at that level. The results suggest that ITS-RFLP does not provide characteristic markers to differentiate B. bassiana isolates recovered from different insect species of various geographical origins.

3

Estimated length (in bp) of digested fragments of the PCR-amplified ITS regions

Isolate IMI number Enzyme 
 HaeIII HpaII AluMob
348083 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
339137 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382806 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382723 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
339140 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
372444 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
351836 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382471 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382807 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
339138 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382294 286, 158, 88, 56, 19 264, 138, 102, 62, 27, 14 496, 111 359, 184, 64 
341545 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382808 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382629 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382763 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382805 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
361056 287, 157, 88, 56, 19 266, 135, 104, 62, 26, 14 607 363, 184, 60 
382809 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382628 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382765 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382764 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
356817 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382626 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382296 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 382, 114, 111 363, 184, 60 
382627 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382470 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382295 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382810 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382630 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
358840 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
327908 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
331275 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
160567 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
334663 286, 158, 88, 56, 19 279, 136, 82, 62, 26, 22 607 184, 183, 180, 60 
129066 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
228343 286, 158, 88, 56, 19 279, 136, 82, 62, 26, 22   
386705 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386706 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386693 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386695 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386696 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386694 287, 158, 88, 56, 19 265, 136, 104, 62, 27, 14 497, 111 363, 185, 60 
386699 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382724 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382302 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386700 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386701 286, 158, 88, 56, 19 264, 138, 102, 62, 16, 14, 11 496, 111 359, 184, 64 
386704 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386703 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
386702 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
Isolate IMI number Enzyme 
 HaeIII HpaII AluMob
348083 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
339137 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382806 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382723 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
339140 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
372444 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
351836 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382471 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382807 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
339138 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382294 286, 158, 88, 56, 19 264, 138, 102, 62, 27, 14 496, 111 359, 184, 64 
341545 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382808 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382629 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382763 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382805 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
361056 287, 157, 88, 56, 19 266, 135, 104, 62, 26, 14 607 363, 184, 60 
382809 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382628 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382765 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382764 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
356817 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382626 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382296 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 382, 114, 111 363, 184, 60 
382627 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382470 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382295 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
382810 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382630 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
358840 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
327908 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
331275 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
160567 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
334663 286, 158, 88, 56, 19 279, 136, 82, 62, 26, 22 607 184, 183, 180, 60 
129066 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
228343 286, 158, 88, 56, 19 279, 136, 82, 62, 26, 22   
386705 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386706 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386693 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386695 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386696 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386694 287, 158, 88, 56, 19 265, 136, 104, 62, 27, 14 497, 111 363, 185, 60 
386699 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
382724 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
382302 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14 496, 111 363, 184, 60 
386700 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386701 286, 158, 88, 56, 19 264, 138, 102, 62, 16, 14, 11 496, 111 359, 184, 64 
386704 286, 158, 88, 56, 19 264, 136, 104, 62, 27, 14   
386703 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 
386702 286, 158, 88, 56, 19 264, 136, 104, 62, 16, 14, 11 496, 111 363, 184, 60 

Genetic Resource Collection, CABI Bioscience, Egham, UK.

None of the isolates had sites for restriction enzymes EcoRI and PstI, length of intact fragments for all isolates was 607 bp, apart from IMI 386694 which was 608 bp.

Chosen isolates for AluI and MobI digest and ITS sequencing.

Fig. 1 shows the neighbour-joining dendrogram for 27 sequences of ITS regions. It shows that the B. bassiana isolates show overall sequence divergence of less than 2.0%. The low variation observed amongst these B. bassiana ITS regions agrees with other literature data (e.g. [25]), and supports the observation that other genetic variants (e.g. virulence-associated loci) are likely to occur more rapidly than the mutations detected in ITS regions.

1

Neighbour-joining tree generated by Clustal W for the 27 sequences of ITS regions, including ITS1-5.8S-ITS2. The sequences have been deposited in the DDBJ, EMBL, and GenBank, and their accession numbers are in parentheses: IMI386702 (AJ560666), IMI386703 (AJ560667), IMI386701 (AJ560668), IMI382302 (AJ560669), IMI382724 (AJ560670), IMI386694 (AJ560671), IMI386696 (AJ560672), IMI386693 (AJ560673), IMI386705 (AJ560674), IMI334663 (AJ560675), IMI382630 (AJ560676), IMI382295 (AJ560677), IMI382470 (AJ560678), IMI382627 (AJ560679), IMI382296 (AJ560680), IMI382626 (AJ560681), IMI356817 (AJ560682), IMI382764 (AJ560683), IMI382765 (AJ560684), IMI382628 (AJ560685), IMI361056 (AJ560686), IMI382763 (AJ560687), IMI382629 (AJ560688), IMI341545 (AJ560689), IMI382294 (AJ560690), IMI382471 (AJ560691), IMI382723 (AJ560692).

1

Neighbour-joining tree generated by Clustal W for the 27 sequences of ITS regions, including ITS1-5.8S-ITS2. The sequences have been deposited in the DDBJ, EMBL, and GenBank, and their accession numbers are in parentheses: IMI386702 (AJ560666), IMI386703 (AJ560667), IMI386701 (AJ560668), IMI382302 (AJ560669), IMI382724 (AJ560670), IMI386694 (AJ560671), IMI386696 (AJ560672), IMI386693 (AJ560673), IMI386705 (AJ560674), IMI334663 (AJ560675), IMI382630 (AJ560676), IMI382295 (AJ560677), IMI382470 (AJ560678), IMI382627 (AJ560679), IMI382296 (AJ560680), IMI382626 (AJ560681), IMI356817 (AJ560682), IMI382764 (AJ560683), IMI382765 (AJ560684), IMI382628 (AJ560685), IMI361056 (AJ560686), IMI382763 (AJ560687), IMI382629 (AJ560688), IMI341545 (AJ560689), IMI382294 (AJ560690), IMI382471 (AJ560691), IMI382723 (AJ560692).

AFLP

Fig. 2 shows two UPGMA (unweighted pair group method with arithmetic mean) dendrograms of relatedness (Dice similarity coefficient) prepared from AFLP banding patterns. The AFLP patterns were compared on a PC using GelCompar II Software (Applied Maths, Kortrijk, Belgium). The dendrograms show the combined results from the three combinations of selective primers, which have nucleotides overhanging at the 3′-end: EcoRI-GC+HpaII-ACC; EcoRI-AT+HpaII-CTC; EcoRI-AT+HpaII-ACC. Fig. 2A shows the results for all 50 isolates used, and Fig. 2B shows the results only for the isolates from Kenya (one B. brongniartii IMI 334663, and 28 B. bassiana).

2

Cluster analysis using band matching and UPGMA. Dendrogram of relatedness (Dice similarity coefficient) prepared from AFLP banding patterns. The AFLP patterns were compared on a PC using GelCompar II Software (Applied Maths, Kortrijk, Belgium). The dendrograms show the combined results from the three combinations of selective primers, which have nucleotides overhanging at the 3′-end: EcoRI-GC+HpaII-ACC; EcoRI-AT+HpaII-CTC; EcoRI-AT+HpaII-ACC. A: All 50 isolates of Beauveria sp., B. brongniartii and B. bassiana used in this study. B: Only isolates from Kenya.

2

Cluster analysis using band matching and UPGMA. Dendrogram of relatedness (Dice similarity coefficient) prepared from AFLP banding patterns. The AFLP patterns were compared on a PC using GelCompar II Software (Applied Maths, Kortrijk, Belgium). The dendrograms show the combined results from the three combinations of selective primers, which have nucleotides overhanging at the 3′-end: EcoRI-GC+HpaII-ACC; EcoRI-AT+HpaII-CTC; EcoRI-AT+HpaII-ACC. A: All 50 isolates of Beauveria sp., B. brongniartii and B. bassiana used in this study. B: Only isolates from Kenya.

The high sensitivity of the AFLP technique and its rich banding pattern (50–100 bands for each pair of selective primers) provided more information on polymorphism between B. bassiana isolates, allowing them to be clustered based on % similarity reflecting their intraspecific genetic relatedness.

Fig. 2A shows that B. bassiana isolates from Papua New Guinea (IMI 129066) and Philippines (IMI 386693) clustered well together with 86% similarity, likewise the isolates from Israel (IMI 339138), Brazil (IMI 341545), Kenya (IMI 382765) and Uganda (IMI 372444) clustered together at 85–80% similarity, and the isolates from Kenya (IMI 327908) and Zimbabwe (IMI 386699) clustered at 81% similarity. These percentages values are not absolute because they depend on the software optimisation and tolerance settings used. In this study, we used band matching and 1.0% tolerance, and we could say that high percentages of similarity (>80%) indicate that the isolates are very closely related to each other. We have observed (data not shown) that repeats of different DNA extracts from the same isolate share more than 90% similarity.

The UPGMA analysis demonstrated that some B. bassiana isolates from distinct geographical origins and various hosts showed little genetic variability. The B. bassiana isolates from Morocco (IMI 351836) and India (IMI 356817), which shared 83% similarity, were placed within a cluster of isolates from Kenya. The B. bassiana isolates IMI 386706 and IMI 386696 are both from Brazil and although they shared 82% similarity, they are from very distant regions (Mato Grosso and Amazonas, respectively) and they were isolated from different insects (Rommatocerus schistocercoides and Diabrotica sp., respectively).

However, the B. bassiana isolates from England (IMI 358840), Kenya (IMI 382810), Costa Rica (IMI 361056), Portugal (IMI 386694) and Colombia (IMI 339140) showed greater genetic variability with lower relative similarity scores ranging from 77 to 63%.

Fig. 2B shows only the isolates from Kenya. B. bassiana IMI 382626 and IMI 382763 shared as much as 89% similarity, although they were recovered from distinct districts in Kenya (Uasin Guishu and Bondo), suggesting that the distinct geographical regions within a country are not a constraint in the relatedness between the isolates.

The IMI 382763 was originally isolated from a Sitophilus zeamais cadaver (IMI 382471) and later re-isolated after passage through Tribolium sp., and it appeared to be closer to an isolate from a distinct geographical region rather than to the original IMI 382471. On the other hand, IMI 382628 remained closely related to IMI 382471 (83% similarity) even after passage through a different host. Another closely related pair of isolates even after re-isolation through pathogenicity test was IMI 382806 and IMI 382808 sharing 84% similarity. Interestingly the isolates IMI 382701 and IMI 386700 which shared 88% similarity were recovered from different insects through pathogenicity test, and became considerably distant from the original IMI 386807. These results suggest that one or more passages through different insects can sometimes play a significant a role on the genetic variability between closely related isolates.

Fig. 2B also suggests a clonal correlation between isolates and host since various clusters of B. bassiana from S. zeamais shared more than 80% similarity, although they were recovered from distinct districts in Kenya. The various S. zeamais clusters correlated to each other at lower relative % similarities, revealing the polymorphism between clonal populations.

The Beauveria sp. isolate (IMI 331275) was comfortably placed amongst all the B. bassiana, while the two B. brongniartii isolates (IMI 334663 and IMI 228343) were placed at lower values of 62% and 58% similarity, respectively, to all the other B. bassiana (Fig. 2A) and 63% similarity comparing to the isolates from Kenya (Fig. 2B). In contrast to the ITS-RFLP technique, the AFLP technique was successful on differentiating the two B. brongniartii isolates from B. bassiana, and it would be a more suitable technique for interspecific identification of isolates.

In conclusion, the adapted AFLP technique described in this paper was reproducible to analyse genomic DNA from Beauveria spp. and effective to show interspecific and intraspecific molecular variability between isolates. Although there was no obvious correlation between isolates, host and geographical origin, the technique revealed clonal populations of B. bassiana isolates used in this study, obtained from S. zeamais from distinct geographical regions within Kenya.

Acknowledgements

The financial support from the UK Department for Environment, Food and Rural Affairs (DEFRA) is gratefully acknowledged.

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