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Phylogenetics of Entomopathogenic Fungi Isolated Paecilomyces spp. (Ascomycota) From the Soils of Different Ecosystems.

Byline: Shoaib Freed, Feng-Liang Jin and Shun-Xiang Ren

Abstract

The entomopathogenic fungi Paecilomyces spp were isolated from the soils of diverse sources i.e., forest, agricultural and urban lands belonging to different geographical locations. The phylogenetic analysis of the ITS1, ITS2 and 5.8S- rDNA sequences of entomopathogenic Paecilomyces spp. from different countries support the polyphyletic character. In the Hypocreales, four key ITS subgroups were found, i.e., Isaria fumosoroseaus, Paecilomyces marquandii, Paecilomyces lilacinus and Paecilomyces sp. All of the four foremost subgroups of the Hypocreales showed clear identification and division. One of the main subgroup showed distinctive characteristics as it showed the majority of P. marquandii, P. lilacinus and Paecilomyces sp. distant relatedness.

In addition the genetic variability among the isolates of I. fumosoroseaus was assessed and compared with the strains from other countries; overall genetic variability among the isolates of different countries was observed that confirms the polymorphicnature of I. fumosoroseaus. The majority of the isolates of I. fumosoroseaus belonged to one of the closely related genotypes. The utmost genetic distance of 0.169% was observed between the strains of France and USA. This information provides an enhanced perceptive concerning the isolates of diverse countries with soil source.

Key words: Molecular phylogeny, Paecilomyces spp, molecular phylogeny, ITS -r-DNA, Isaria fumosorosea,us biocontrol, mycoinsecticide.

INTRODUCTION

Fungi play an imperative physiological, meditative and ecological role in the ecosystem (Miller, 1995). Soil is a multifaceted ecosystem in which micro-organisms occur in heterogeneous communities; however, the behaviour of individual species is often unknown due to the lack of appropriate detection and identification techniques (Akkermans et al., 1994). The hypomycete genus Paecilomyces contains members which are often thermophilic; the section Isarioidea contains mesophiles, including numerous entomopathogenic species such as IsariaPaecilomyces farinosus, Isaria fumosoroseaeus, Paecilomyces amoeneroseus, Paecilomyces lilacinus, Paecilomyces javanicus and Paecilomyces tenuipes. For the control of insect pests especially Bemisia tabaci (Gennadius), considered as a major pest in field and green house crops, I. fumosoroseaus is regarded as the most common pathogen (Laecey et al., 1996).

The release of I. fumosorosea Paecilomyces spp. in the field for the effective control of insect pests needs the selection of best control of insect pests needs the selection of best genotypes and for this the study of population genetics becomes the prerequisite (Clouet et al., 2005). The limitations of the traditional identification techniques indicate that non conventional methods need to be developed for the identification of these fungi (Inglis and Tigano, 2006).

Comparative RNA (rDNA) gene sequence information contain a tandem repeat including codon regions which are conserved at varying degrees and are also highly divergent due to this reason these give an enhanced knowledge about the evolutionary interaction over a broad range ( Pace et al., 1986; Driver et al., 2000). Luangsa-ard et al. (2004, 2005) examined the nuclear-encoded SSR DNA sequences of Paecilomyces strains from Thailand and some of others held in CBS. The phylogenetic analysis based on the 18S -r-DNA demonstrated that Paecilomyces is polyphyletic across two subclasses; subsequently the ss-tubulin gene and ITS -r-DNA sequences analysis confirmed the phylogenetic relationship of Paecilomyces sect.

Isarioidea and it showed that this section is also polyphyletic within the Hypocreales (Luangsa-ard et al., 2005). In case for the Paecilomyces the analysis of large and small subunit rRNA gene, ITS-rDNA and especially for I. fumosoroseaus has already been performed. (Obornik et al., 2001; Fargues et al., 2002). The genetic variability of I. fumosoroseaus was characterized by several methods, 28S-rDNA genes, RAPD-PCR and tRNA-PCR showed polymorphism in the isolates of I. fumosoroseaus (Tigano-Milani et al., 1995a; Fegan et al., 1993; Leal et al., 1994; Bidochka et al., 1994; Piatti et al., 1998).

In this study we isolated and selected various Paecilomyces spp. from the soil samples of different geological origins and sources to clarify the taxonomic relationships among them and to uncover the genetic variability among the isolates of I. fumosoroseaus isolates from different countries with diverse sources of isolation.

MATERIALS AND METHODS

Sampling locations

The soil samples from a variety of habitats including urban, agricultural and forest ecosystems were collected from Islamic Republic of Pakistan, People's Republic of. R. China, South Korea, Indonesia and The Netherlands. Soil samples were taken 10 cm deep under the earth from different provinces of the above described countries. After the collection, samples were preserved at 4degC untill used for isolation of Isaria and Paecilomyces spp.

Isolation of fungi

For soil samples collected from different locations, Isaria and Paecilomyces spp. were isolated on semi-selective medium described by (Veen and Ferron, 1966). The medium consisted of glucose (10 g), peptone (10 g), bile (15 g) (Sigma), rose Bengal (1:15,000), and agar (30 g) in 1 L of deionized water. The medium was amended with 0.002 gL-1 dodine (N-dodecylguanidine monoacetate; American Cyanamid), 0.12 g L-1 cycloheximide (Sigma) and 0.25 gL-1 chloramphenicol (Sigma). Sub-sample of soil (10g) was placed into 90 ml of sterile water, and the sample was homogenized using a surface-sanitized waring blender. The homogenate was diluted three times in a 10-fold dilution series in sterile phosphate buffer (100 mM, pH7.0), 100 ul from each dilution was spread onto the semi-selective medium, and cultures were maintained at 25degC in the dark.

Cultures were examined after 4 days and daily thereafter for 3 additional days. Conidia characteristicharacteristiccs of Isaria and Paecilomyces spp. were transferred onto PDA, thecultures were purified, and examined microscopically for characteristic conidiogenesis.

All isolates were subsequently propagated from a single conidium (Table I). 100 ul of a suspension containing a low density of conidia in phosphate buffer was spread onto PDA. After 24 h growth at 25degC, a single germinated conidium separated from other conidia was identified by dissecting microscope using 20 times magnification, a piece of the medium encompassing only the target conidium was aseptically removed and transferred to PDA, and the culture was maintained at room temperature. After 5 days, morphological characteristics of Isaria and Paecilomyces spp. were confirmed microscopically; conidia were collected and stored in sterile 30 % glycerol at -80degC until required.

Genomic DNA isolation

The genomic DNA was extracted by the method described by Liu et al. (2000) with some modifications. Briefly, the method included the following steps (i) To a 1.5-ml Eppendorf tube containing 500 ul of lysis buffer (400mM Tris-HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate), a small lump of mycelia was added by using a sterile toothpick, with which the lump of mycelia was disrupted.

The tube was then left at room temperature for 45 min. (ii) After adding 150 ul of potassium acetate (pH 4.8; made of 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of distilled water), the tube was then vortexed briefly and spun at greater than10,000 x g for 2 min. (iii) The supernatant was transferred to another 1.5 -ml Eppendorf tube and centrifuged again as described above. After transferring the supernatant to a new 1.5 -ml Eppendorf tube, an equal volume of isopropyl alcohol was added. The tube was mixed by inversion briefly. (iv) The tube was spun at 10,000 x g for 2 min, while he supernatant was discarded.

The resultant DNA pellet was washed twice with 300 ul of 70% ethanol. After the pellet was spun at greater than 10,000 rpm for 1 min, the supernatant was discarded. The DNA pellet was air dried and dissolved in 70 ul of 1 x Tris-EDTA.

Extracted genomic DNA was identified by 1% TBE-agarose gel (Invitrogen Corp., Burlington, ON) electrophoresis, stained with ethidium bromide and visualized under UV light. The DNA was purified by using the E.Z.N.A. Gel Extraction Kit (OMEGA bio-tek, USA). The purified DNA was used for the PCR amplification.

Table I.- Entomopathogenic fungal isolates from different geographical origin and source

S.No.###Isolates###Fungal species###Origin###Province/City###Source###Accession numbers

1.###CNIM###Isaria fumosorosea###P.R.China###Inner Mongolia###Riverside soil###FJ765006

2.###CNZG###Isaria fumosorosea###P.R.China###Guangdong,Zhanjiang###Farm land soil###FJ765007

3.###CNZH###Isaria fumosorosea###P.R.China###Zhejiang Ningbo###Mountain soil###FJ765008

4.###CNHG###Isaria fumosorosea###P.R.China###Gansu Huixian###Farm land soil###FJ765009

5.###CNBJ###Isaria fumosorosea###P.R.China###Beijing###Urban land soil###FJ765010

6.###CNXN###Isaria fumosorosea###P.R.China###Xinjiang###Forest soil###FJ765011

7.###NLHG-1###Isaria fumosorosea###Netherlands###Gelderland, Heteren###Vegetable field soil###FJ765012

8.###NLHG-2###Isaria fumosorosea###Netherlands###Gelderland, Heteren###Corn field soil###FJ765013

9.###NLWG###Isaria fumosorosea###Netherlands###Gelderland, Wageningen Grass field soil###FJ765014

10.###NLUC###Isaria fumosorosea###Netherlands###Utrecht###Urban land soil###FJ765015

11.###NLAG###Isaria fumosorosea###Netherlands###Gelderland, Arnhem###Potato field soil###FJ765016

12.###SKCH-1###Isaria fumosorosea###South Korea###Cheju island###Urban land soil###FJ765017

13.###SKCH-2###Paecilomyces sp.###South Korea###Cheju island###Seaside soil###FJ765018

14.###CNXM###Paecilomyces. lilacinus###P.R.China###Xizang, Motuo###Forest soil###FJ765019

15.###SKCH-4###Paecilomyces. lilacinus###South Korea###Cheju island###Urban land soil###FJ765020

16.###PAK-1###Paecilomyces. lilacinus###I.R.Pakistan###Rawlakot, Kashmir###Mountain soil###FJ765021

17.###PAK-2###Paecilomyces. lilacinus###I.R.Pakistan###Muzaffarabad, Kashmir###Mountain soil###FJ765022

18.###INTR-1###Paecilomyces. lilacinus###Indonesia###Tretes###Mountain soil###FJ765023

19.###INTR-2###Paecilomyces. lilacinus###Indonesia###Tretes###Volcano soil###FJ765024

20.###CNSH###Paecilomyces marquandii###P.R.China###Hubei, Shiyan###Mountain soil###FJ765025

21.###SKCH-5###Paecilomyces marquandii###South Korea###Cheju Island###Volcano soil###FJ765026

22.###CNLH###Penicilium citrinum###P.R.China###Hubei, Liuyang###Mountain soil###FJ765031

PCR amplification for ITS-rDNA regions

DNA was amplified by PCR using the complementary primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4A (5'-GCCGTTACTGGGGCAATCCCTG-3') Larena et al. (1999). The PCR mixture consisted of a total volume of 25ul containing 1 x reaction buffer, 0.2mM dNTPs, 2mM MgCl2, 0.5uM of each primer (Invitrogen , USA), and 1U Taq polymerase (TAKARA Japan) and 2.0ul (20-25 ng) of DNA template. The amplification conditions consisted of one cycle at 94degC for 5 min, followed by 35 cycles at 94degC for 30s, 58degC for 1 min and 72degC for 50s, finishing up with 1 cycle at 72degC for 5 min.

DNA visualization, quantification and purification

The PCR products were identified by agarose gel electrophoresis in a 1.5% TAE-agarose gel. Quantification of the PCR products was done by using a 100bp DNA ladder. Products with clear band were purified by using the E.Z.N.A. Gel Extraction Kit (OMEGA bio-tek, USA).

DNA sequencing and data analysis

The purified PCR amplified products were used for nucleotide sequencing. DNA sequencing was done by using the same primers with the concentrations as described above. Nucleotide sequences for the isolates were searched for their similarity index by using BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Manual improvement was also done to the sequences lacking a few amino acids on their two ends.

This was realized by retrieving the whole subject sequence from GenBank and translating it with EditSeq program (version 5.01) of the DNAStar package to obtain the absent amino acid residues. All the sequences were aligned using Muscle online (www.ebi.ac.uk/Tools/muscle/index.html). Alignment gaps were treated as missing data and no characters in the alignment were excluded. Phylogenetic analyses were conducted on the basis of 658 aligned positions alignable in all sequences. Divergence of each pair of sequence was calculated by DAMBE Xia and Xie (2001).

Table II.- Isaria and Paecilomyces spp. isolates (ITS-rDNA) sequences downloaded from GenBank used in the phylogenetic tree

S.No.###Accession No's###Species###Location

1.###AB265146###Isaria fumosorosea###Japan

2.###AF461747###Isaria fumosorosea###France

3.###AF461748###Isaria fumosorosea###France

4.###AJ345091###Isaria fumosorosea###Hungary

5.###AJ345092###Isaria fumosorosea###Hungary

6.###AJ608982###Isaria fumosorosea###United Kingdom

7.###AY624182###Isaria fumosorosea###Thailand

8.###AY755506###Isaria fumosorosea###Germany

9.###DQ069285###Isaria fumosorosea###Indonesia

10.###DQ449655###Isaria fumosorosea###Spain

11.###EU886747###Isaria fumosorosea###USA

12.###EU886748###Isaria fumosorosea###USA

13.###AM412779###Paecilomyces lilacinus###Germany

14.###AY213668###Paecilomyces lilacinus###USA

15.###EU306174###Paecilomyces lilacinus###Taiwan

16.###EU828665###Paecilomyces lilacinus###Thailand

17.###AB369489###Paecilomyces lilacinus###Gansu, P.R. China

18.###FJ461773###Paecilomyces lilacinus###Hunan, P.R.China

19.###AY624193###Paecilomyces marquandii###Thailand

20.###AB244776###Paecilomyces marquandii###Japan

Phylogenetic analyses were conducted using PAUP 4.0 Beta Win (Swofford, 1998). The aligned sequences were used to construct the maximum parsimony tree. Sequences were first aligned in Muscle and then copied into PAUP window to prepare nexus files. Maximum parsimony trees were bootstrapped with 1,000 replicates to provide information about their statistical reliability. Maximum parsimony trees were constructed using PAUP 4.0 Beta Win by executing command "bootstrap nreps = 100 search = heuristic/addseq = random". Other parameters were set to default values.

Relative support for resulting trees was obtained from bootstrap analyses (Felsenstein, 1985) using 100 heuristic searches with groups occurring at 70% or greater frequencies being retained in the consensus trees. Maximum likelihood trees were constructed using MrBayes 3 (Ronquist and Huelsenbeck, 2003). The trees were displayed using the TreeView program (version 1.6.6) (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html).

RESULTS

Taxonomic relationships of Isaria and Paecilomyces spp. isolated from different agricultural, forest and urban land soil of Pakistan, China, South Korea, Indonesia and The Netherlands was assessed. The ITS-1, ITS-2 and 5.8S- rRNA genes were amplified and nucleotide sequencing was performed. The representative nucleotide sequences for the Isaria and Paecilomyces spp. strains were downloaded from GenBank and were clustered with the laboratory isolated Isaria and Paecilomyces spp. The maximum parsimony and maximum likelihood methods generated somewhat similar dendrograms, therefore only the dendrograms of maximum likelihood have been included in the results. The dendogram obtained by maximum likelihood (ML) method using MrBayes 3.0, phylogenetic software clustered all of the isolates into 3 main groups with certain sub groups (Fig. 1). The group A clustered I. fumosoroseaus isolates from different countries.

The clade 1-A clustered the isolates of laboratory isolated I. fumosoroseaus, while the clade 1-B groupe together the isolates of Iof I. fumosoroseaus from different countries viz., USA, Thailand, Germany etc. The genetic variability among all the isolates of I. fumosoroseaus used in the study was found to be minimal, even with different geographical origins from all the isolates used in the study.

The Group B clustered P. lilacinus and Paecilomyces sp. isolates from different geographical origins. The grouping of P. lilacinus from Asia, Europe and America, demonstrated the results somewhat similar to that of group-A, as it illustrated same kind of diversification i.e., the clustering of isolates from Asia, Europe and USA. In contrast to this P. marquandii from different geographical origins (Asian countries i.e., China, South Korea, Thailand and Japan) was grouped in clade C. The clustering of all the isolates of P. marquandii shows the grouping and its difference in genetic make up from that of other Paecilomyces spp. The dendogram obtained by ML method clearly indicates the presence of four main sub groups of Paecilomyces i.e., I. fumosoroseaus, P. marquandii, P. lilacinus and Paecilomyces sp. of the order Hypocreales of Ascomycota, while outgroup consisted of Eurotiales (Fig. 1).

The isolates of P. lilacinus and Paecilomyces sp. showed a basal clade and the presence of these species together e group shows that some characters are being shared by both species.

Genetic variability of I. fumosoroseaus

The overall genetic variability among the laboratory isolated I. fumosoroseaus isolates was determined (Fig. 2). The dendogram obtained by ML analysis clustered the isolates into two main groups. The group-A clustered mainly isolates from Europe, UK and USA while other lab isolates viz., CNIM, CNHG, CNZH and CNXJ didn't cluster in this group. Group-B assembled mainly the laboratory isolated samples and in addition it also grouped together few isolates from France, Germany and Indonesia. The genetic variability among the isolates seemed to be minimal, the reason for which can be the sharing of the same host.

Genetic distances of I. fumosoroseaus isolates according to locations

The genetic distances among the isolates I. fumosoroseaus from different ecosystems were calculated by Kimura 2-parameter model. The results showed different genetic distances for the isolates of I. fumosoroseaus isolates. The genetic distances among the isolates range from 0.000 to 0.169%. The maximum genetic distance of 0.169% was observed between the isolates of France and USA.

DISCUSSION

Phylogenetic analysis based on the ITS-rDNA regions revealed a high level of polymorphism within the isolates of I. fumosoroseaus and other related Paecilomyces species. Four distinct groups of Paecilomyces spp. were observed from the ITS-rDNA sequence data. All I. fumosoroseaus isolates were clustered in group-A. The second group i.e. B, clustered P. lilacinus while the third group C, contained P. marquandii from different countries (Table I) (Fig. 1).

The molecular analysis of the Paecilomyces spp. demonstrates that the insect associated Paecilomyces are polyphyletic within the order Hypocreales (Fig. 1). The order Hypocreales was clearly divided into different sub groups as the group-A contained the entomopathogenic fungi I. fumosoroseaus, while the presence of the nematophagous fungi P. lilacinus in group-B proved the findings of Luangsa-ard et al. (2005) that the section Isarioidea of the order Hypocreales is not monophyletic. P. lilacinus isolates were grouped together with another related species i.e.,Paecilomyces sp. that confirms the relationship of the order Hypocreales with its subgroups. In contrast to this P. marquandii was clustered group-C that shows the division of Paecilomyces groups.

The ITS-rDNA sequence data showed a high level of variability that divided the three species of Paecilomyces into four groups i.e., I. fumosoroseaus, P. marquandii, P. lilacinus and Paecilomyces sp. Our results are in confirmation with the results of Inglis and Tigano (2006), that a high level of variability was observed among the Paecilomyces spp. isolates from different countries and hosts. The same results were obtained by using different DNA markers (Tigano-Milani et al., 1995a, b). The isolates of Paecilomyces spp. were clustered into three distinct groups while the genetic variability among the isolates of the same Paecilomyces spp. seemed to be minimal, even the isolates were isolated from different geographical locations and hosts (Fig. 1). The group A

confers comprehensive detail regarding the relationship of I. fumosoroseaus, as it clustered together the isolates from three different continents viz., Asia, Europe and America.

The maximum genetic distance of 0.169% was observed between the strains of I. fumosoroseaus from France and USA. It can be assumed that the entomopathogenic fungis possess little genetic variability as in case of Metarhizium anisopliae var. anisopliae (Freed et al., 2010; Inglis et al., 2008). The ITS region was found to be useful for resolving the difficulties in the taxonomy of Paecilomyces spp. The nucleotide sequences of different Paecilomyces spp. were downloaded from GenBank for their relationship with sequence data from our study. In this case alsofurthermore the same results were obtained. All the nucleotide sequences together with laboratory isolates were clustered into 3 main groups with sub groups This suggests that Paecilomyces spp. are closely related to each other and also with other entomopathogenic fungi as reported by Obornik et al. (2001).

Previous phylogenetic studies on I. fumosoroseaus showed that there are at least three monophyletic groups within this complex and that host selection pressure may be significant in the selection of genotypes (Fargues et al., 2002), as it is significant mycoinsecticide for the control of B. tabaci (Laecey et al., 1996; 2001). Our studies showed that I. fumosoroseaus isolates were only clustered with the genetically identical I. fumosoroseaus isolates, none of the other Paecilomyces spp. isolates were clustered with these isolates (Fig. 2). This gives strong clueevidence that these isolates are genetically different from the other Paecilomyces spp. isolates and the less genetic variability among these isolates suggestproposess that it maymay possibly be due to the same source of isolation i.e., soil sources, while the other isolates used in previous studies were isolated from the insect hosts i.e., B. tabaci (Tignao-Milani et al., 1995a; Cantone and Vandenberg, 1998).

Our studies confirm the results as that of Luangsa-ard et al. (2004), studying the large and small subunits rRNA gene sequences, where Paecilomyces probably represents a genus only. The molecular results based on the nucleotide sequences of ITS-rDNA region support the previous studies of Paecilomyces spp. and will helpfacilitate for the further studies related to the phylogenetics of Paecilomyces spp.

ACKNOWLEDGMENTS

This research was funded by a grant from the National Natural Science Foundation of China (31071685)Basic Research and Development Program ,of China 973 Project (2006CB102005), the Special Scientific Research Fund for Commonwealth Trade of China (200803005).

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(Received 10 January 2010, , revised 15 October2 November 2010)

Engineering Research Center of Biocontrol, Ministry of Education, South China Agricultural University, Guangzhou, 510642, P.R.China
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Author:Freed, Shoaib; Jin, Feng-Liang; Ren, Shun-Xiang
Publication:Pakistan Journal of Zoology
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Geographic Code:9CHIN
Date:Jun 30, 2011
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