Environment Health Techniques Bacterial associated with Meloidogyne incognita
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Research Paper Associated bacteria of different life stages of Meloidogyne incognita using pyrosequencing-based analysis Yi Cao1,2, Baoyu Tian3, Xinglai Ji1, Shenghua Shang2, Chaojun Lu1 and Keqin Zhang1 1
2 3
Key Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China Key Laboratory of Molecular Genetics, Guizhou Academy of Tobacco Science, Guiyang, China College of Life Science, Fujian Normal University, Fuzhou, China
The root knot nematode (RKN), Meloidogyne incognita, belongs to the most damaging plant pathogens worldwide, and is able to infect almost all cultivated plants, like tomato. Recent research supports the hypothesis that bacteria often associated with plant-parasitic nematodes, function as nematode parasites, symbionts, or commensal organisms etc. In this study, we explored the bacterial consortia associated with M. incognita at different developmental stages, including egg mass, adult female and second-stage juvenile using the pyrosequencing approach. The results showed that Proteobacteria, with a proportion of 71–84%, is the most abundant phylum associated with M. incognita in infected tomato roots, followed by Actinobacteria, Bacteroidetes, Firmicutes etc. Egg mass, female and second-stage juvenile of M. incognita harbored a core microbiome with minor difference in communities and diversities. Several bacteria genera identified in M. incognita are recognized cellulosic microorganisms, pathogenic bacteria, nitrogenfixing bacteria and antagonists to M. incognita. Some genera previously identified in other plantparasitic nematodes were also found in tomato RKNs. The potential biological control microorganisms, including the known bacterial pathogens and nematode antagonists, such as Actinomycetes and Pseudomonas, showed the largest diversity and proportion in egg mass, and dramatically decreased in second-stage juvenile and female of M. incognita. This is the first comprehensive report of bacterial flora associated with the RKN identified by pyrosequencingbased analysis. The results provide valuable information for understanding nematode–microbiota interactions and may be helpful in the development of novel nematode-control strategies. Keywords: Meloidogyne incognita / Biological control / Nematode-associated bacterial consortia / Microbial diversity / Pyrosequencing Received: October 23, 2014; accepted: January 30, 2015 DOI 10.1002/jobm.201400816
Introduction Nematode-bacterium associations are considered one of the best models to investigate microbe–host interactions, partly due to the well-studied model organism Caenorhabditis elegans, and the broad association in bacterial and nematode communities in various environments, such as marine, soil, animal or plant host [1]. Bacteria can Correspondence: Keqin Zhang, Key Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, No. 2, North Green Lake Road, Wuhua District, Kunming 650091, Yunnan, China E-mail: [email protected] Phone: þ86 0871 65034878 Fax: þ86 0871 65034878 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
be parasites, symbionts, commensal organisms or a food source for the nematode hosts. For instance, thixotropic surface-colonizing bacteria are located on the outside of Laxus oneistus marine nematodes as symbionts [2]. Several microorganisms inhabit the gut of the bacteriovorus free-living soil nematode Acrobeloides maximus [3]. The endosymbiont Wolbachia co-evolved with filarial nematodes and is critical for host development, fertility, and viability. This relationship has been exploited in treatment of the widespread filarial disease [4–6]. Xenorhabdus and Photorhabdus are proposed to be necessary to the life cycle of the entomopathogenic nematodes Steinernema and Heterorhabditis [7]. In plant parasitic nematodes, the bacterial community associated with the pinewood
www.jbm-journal.com
J. Basic Microbiol. 2015, 54, 1–11
2
Yi Cao et al.
nematodes (PWN) Bursaphelenchus xylophilus and B. mucronatus was characterized by culture-dependent and culture-independent methods [8–11]. The PWN microbiome may contribute to the virulence [12, 13], growth [14], reproduction [15] and xenobiotic detoxification of the nematode [16]. Furthermore, diverse bacteria have been identified to be associated with the cyst nematode Heterodera glycines [17, 18], burrowing nematodes Radopholus similis [19], and dagger nematodes Xiphinema americanum [20]. Genomic analysis of root knot nematodes Meloidogyne incognita and M. halpha revealed that certain nematode genes have been derived from bacteria by horizontal gene transfer (LGT) [21, 22], which has played an important role in the evolution of parasitism [23, 24]. Root knot nematodes belong to the genus Meloidogyne and form one of the most common and destructive groups of plant-parasitic nematodes. The Southern root knot nematode M. incognita, a major species in this genus, is an obligatory biotrophic pathogen which feeds on the roots of almost all cultivated plants, resulting in devastating adverse effects on the crop quality and yield [25]. The life cycle of M. incognita (Fig. 1) contains two phases: the endophytic phase in the root and the exophytic phase in the soil. During the exophytic phase, the nematodes hatch from egg masses on the root surface
Figure 1. The life cycle of Meloidogyne incognita. The females live inside the root where they feeds, lay eggs into gelatinous masses mainly composed of glycoproteins matrix on the root surface, which are called egg masses. Embryogenesis proceeds to the first-stage juvenile (J1) within the egg and then moults to the second-stage juvenile (J2) when conditions are favorable. Hatched J2 invade host plant root in the zone of elongation, after entering the roots they induce the formation of a permanent feeding site and become sedentary. The J2s undergo three further molts to develop from the third-stage juvenile (J3), then the fourth-stage juvenile (J4) and finally into the adult stage. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
as second-stage juveniles (J2) and then invade the plant roots. After burrowing into the host root, the J2s undergo three molts to develop into the adult stage [26]. Despite their economic importance in agriculture, there are few successful methods of controlling these prominent invasive pests. A broad survey of the bacterial diversity associated with RKN may provide valuable information for understanding nematode–microbe interactions and developing novel management strategies [27, 28]. The recent advent of next generation sequencing provides new, cost-efficient and fast strategies to assess the diversity of microbial communities with higher resolution [29]. In the present study, high-throughput tagencoded FLX amplicon pyrosequencing was used to characterize bacterial communities associated with three life stages (Egg mass, adult female and J2) of M. incognita.
Materials and methods Nematodes samples preparation and DNA extraction M. incognita race 1 was kindly provided by China Agricultural University. The nematodes were maintained on susceptible tomato (Solanum lycopersium cv. Jiabao) under greenhouse conditions. Plant roots were harvested from infected tomato plants (55 days after inoculating with single egg masses at the fourth true leaf stage) according to the previously described procedure [30]. Loose soil was manually removed from the roots by kneading and shaking with sterile gloves. Root pieces with galls were washed in running tap water for 2–4 min and then washed several times with sterile water to remove soil. Egg masses of similar sizes were picked with sterilized needles from the roots. The pear-shaped adult females were carefully handpicked with sterilized forceps from dissected roots under a dissection microscope. Second-stage Juveniles (J2s) were hatched from some of the egg masses using a modified method: egg masses were treated with sodium hypochlorite (0.5% v/v) for 30 s, followed by deposition on a sterile 25 mm sieve, washed thoroughly with sterile water, then placed in sterile distilled water at 25 °C to hatch. The hatched J2s were used at less than three days of age. Foreign bacteria, such as soil and plant endophytic bacteria, which loosely associated with the nematodes were removed by surface sterilization in a modified method as previously described [17]. Nematode samples were soaked for 5 min in sodium hypochlorite (0.5% v/v), followed by washing five times with sterile distilled water. Contamination was tested by dropping 50 mL of treated nematode suspension onto nutrient agar (NA) plates, which were incubated for 24 h. For further analysis only nematode samples with no
www.jbm-journal.com
J. Basic Microbiol. 2015, 54, 1–11
Bacterial associated with Meloidogyne incognita
bacterial growth on NA were included. Each sample used for DNA extraction consisted of about 150 egg masses, 500 adult females and 9000 J2s pooled together from 12 infested tomato roots. The pooled nematodes were pelleted and pulverized by three successive freeze-thaw cycles in liquid N2. Metagenomic DNA from adult females, egg masses and J2s was extracted using a Power Soil1 DNA Isolation Kit (MoBio) according to the manufacturer’s instructions. PCR amplification and pyrosequencing DNA fragments of approximately 500 bp of the bacterial 16S rRNA gene, targeting the hypervariable region V1–V3, were amplified using the primer pair 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 533R (50 -TTACCGCGGCTGCTGGCAC-30 ) fused with the 454 Life Sciences primers, barcodes and adapters. The PCR amplifications were carried out in triplicate 50-mL reactions containing 10 ng metagenomic DNA, 2.5U Platinum Taq DNA Polymerase (Invitrogen), 1 mM of each barcoded fusion primer, 0.2 mM of dNTPs in the appropriate 10 PCR Buffer and de-ionized ultrapure water. Negative control samples were treated similarly with the exclusion of template DNA. PCR reactions were started by an initial denaturation at 94 °C for 30 s, followed by five cycles of 94 °C for 20 s, 45 °C for 20 s, 65 °C for 60 s, followed by 20 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s, with a final extension step of 5 min at 72 °C. The PCR products from replicated reactions of the same sample were pooled and purified from 1.5% agarose gels with a QIAquick Gel Extraction Kit (Qiagen). Purified DNA was quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). DNA amplicons were then combined in equimolar ratios into a single tube. Pyrosequencing was performed using the 454 GS FLX platform (Roche). Data processing and bacterial population analysis Data were processed by following the pipelines with MOTHUR [31]. Sequences with ambiguous bases, primer mismatches, homopolymer runs in excess of six bases or errors in barcodes, and with length shorter than 200 bp, or average quality score
1
Research Paper Associated bacteria of different life stages of Meloidogyne incognita using pyrosequencing-based analysis Yi Cao1,2, Baoyu Tian3, Xinglai Ji1, Shenghua Shang2, Chaojun Lu1 and Keqin Zhang1 1
2 3
Key Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China Key Laboratory of Molecular Genetics, Guizhou Academy of Tobacco Science, Guiyang, China College of Life Science, Fujian Normal University, Fuzhou, China
The root knot nematode (RKN), Meloidogyne incognita, belongs to the most damaging plant pathogens worldwide, and is able to infect almost all cultivated plants, like tomato. Recent research supports the hypothesis that bacteria often associated with plant-parasitic nematodes, function as nematode parasites, symbionts, or commensal organisms etc. In this study, we explored the bacterial consortia associated with M. incognita at different developmental stages, including egg mass, adult female and second-stage juvenile using the pyrosequencing approach. The results showed that Proteobacteria, with a proportion of 71–84%, is the most abundant phylum associated with M. incognita in infected tomato roots, followed by Actinobacteria, Bacteroidetes, Firmicutes etc. Egg mass, female and second-stage juvenile of M. incognita harbored a core microbiome with minor difference in communities and diversities. Several bacteria genera identified in M. incognita are recognized cellulosic microorganisms, pathogenic bacteria, nitrogenfixing bacteria and antagonists to M. incognita. Some genera previously identified in other plantparasitic nematodes were also found in tomato RKNs. The potential biological control microorganisms, including the known bacterial pathogens and nematode antagonists, such as Actinomycetes and Pseudomonas, showed the largest diversity and proportion in egg mass, and dramatically decreased in second-stage juvenile and female of M. incognita. This is the first comprehensive report of bacterial flora associated with the RKN identified by pyrosequencingbased analysis. The results provide valuable information for understanding nematode–microbiota interactions and may be helpful in the development of novel nematode-control strategies. Keywords: Meloidogyne incognita / Biological control / Nematode-associated bacterial consortia / Microbial diversity / Pyrosequencing Received: October 23, 2014; accepted: January 30, 2015 DOI 10.1002/jobm.201400816
Introduction Nematode-bacterium associations are considered one of the best models to investigate microbe–host interactions, partly due to the well-studied model organism Caenorhabditis elegans, and the broad association in bacterial and nematode communities in various environments, such as marine, soil, animal or plant host [1]. Bacteria can Correspondence: Keqin Zhang, Key Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, No. 2, North Green Lake Road, Wuhua District, Kunming 650091, Yunnan, China E-mail: [email protected] Phone: þ86 0871 65034878 Fax: þ86 0871 65034878 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
be parasites, symbionts, commensal organisms or a food source for the nematode hosts. For instance, thixotropic surface-colonizing bacteria are located on the outside of Laxus oneistus marine nematodes as symbionts [2]. Several microorganisms inhabit the gut of the bacteriovorus free-living soil nematode Acrobeloides maximus [3]. The endosymbiont Wolbachia co-evolved with filarial nematodes and is critical for host development, fertility, and viability. This relationship has been exploited in treatment of the widespread filarial disease [4–6]. Xenorhabdus and Photorhabdus are proposed to be necessary to the life cycle of the entomopathogenic nematodes Steinernema and Heterorhabditis [7]. In plant parasitic nematodes, the bacterial community associated with the pinewood
www.jbm-journal.com
J. Basic Microbiol. 2015, 54, 1–11
2
Yi Cao et al.
nematodes (PWN) Bursaphelenchus xylophilus and B. mucronatus was characterized by culture-dependent and culture-independent methods [8–11]. The PWN microbiome may contribute to the virulence [12, 13], growth [14], reproduction [15] and xenobiotic detoxification of the nematode [16]. Furthermore, diverse bacteria have been identified to be associated with the cyst nematode Heterodera glycines [17, 18], burrowing nematodes Radopholus similis [19], and dagger nematodes Xiphinema americanum [20]. Genomic analysis of root knot nematodes Meloidogyne incognita and M. halpha revealed that certain nematode genes have been derived from bacteria by horizontal gene transfer (LGT) [21, 22], which has played an important role in the evolution of parasitism [23, 24]. Root knot nematodes belong to the genus Meloidogyne and form one of the most common and destructive groups of plant-parasitic nematodes. The Southern root knot nematode M. incognita, a major species in this genus, is an obligatory biotrophic pathogen which feeds on the roots of almost all cultivated plants, resulting in devastating adverse effects on the crop quality and yield [25]. The life cycle of M. incognita (Fig. 1) contains two phases: the endophytic phase in the root and the exophytic phase in the soil. During the exophytic phase, the nematodes hatch from egg masses on the root surface
Figure 1. The life cycle of Meloidogyne incognita. The females live inside the root where they feeds, lay eggs into gelatinous masses mainly composed of glycoproteins matrix on the root surface, which are called egg masses. Embryogenesis proceeds to the first-stage juvenile (J1) within the egg and then moults to the second-stage juvenile (J2) when conditions are favorable. Hatched J2 invade host plant root in the zone of elongation, after entering the roots they induce the formation of a permanent feeding site and become sedentary. The J2s undergo three further molts to develop from the third-stage juvenile (J3), then the fourth-stage juvenile (J4) and finally into the adult stage. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
as second-stage juveniles (J2) and then invade the plant roots. After burrowing into the host root, the J2s undergo three molts to develop into the adult stage [26]. Despite their economic importance in agriculture, there are few successful methods of controlling these prominent invasive pests. A broad survey of the bacterial diversity associated with RKN may provide valuable information for understanding nematode–microbe interactions and developing novel management strategies [27, 28]. The recent advent of next generation sequencing provides new, cost-efficient and fast strategies to assess the diversity of microbial communities with higher resolution [29]. In the present study, high-throughput tagencoded FLX amplicon pyrosequencing was used to characterize bacterial communities associated with three life stages (Egg mass, adult female and J2) of M. incognita.
Materials and methods Nematodes samples preparation and DNA extraction M. incognita race 1 was kindly provided by China Agricultural University. The nematodes were maintained on susceptible tomato (Solanum lycopersium cv. Jiabao) under greenhouse conditions. Plant roots were harvested from infected tomato plants (55 days after inoculating with single egg masses at the fourth true leaf stage) according to the previously described procedure [30]. Loose soil was manually removed from the roots by kneading and shaking with sterile gloves. Root pieces with galls were washed in running tap water for 2–4 min and then washed several times with sterile water to remove soil. Egg masses of similar sizes were picked with sterilized needles from the roots. The pear-shaped adult females were carefully handpicked with sterilized forceps from dissected roots under a dissection microscope. Second-stage Juveniles (J2s) were hatched from some of the egg masses using a modified method: egg masses were treated with sodium hypochlorite (0.5% v/v) for 30 s, followed by deposition on a sterile 25 mm sieve, washed thoroughly with sterile water, then placed in sterile distilled water at 25 °C to hatch. The hatched J2s were used at less than three days of age. Foreign bacteria, such as soil and plant endophytic bacteria, which loosely associated with the nematodes were removed by surface sterilization in a modified method as previously described [17]. Nematode samples were soaked for 5 min in sodium hypochlorite (0.5% v/v), followed by washing five times with sterile distilled water. Contamination was tested by dropping 50 mL of treated nematode suspension onto nutrient agar (NA) plates, which were incubated for 24 h. For further analysis only nematode samples with no
www.jbm-journal.com
J. Basic Microbiol. 2015, 54, 1–11
Bacterial associated with Meloidogyne incognita
bacterial growth on NA were included. Each sample used for DNA extraction consisted of about 150 egg masses, 500 adult females and 9000 J2s pooled together from 12 infested tomato roots. The pooled nematodes were pelleted and pulverized by three successive freeze-thaw cycles in liquid N2. Metagenomic DNA from adult females, egg masses and J2s was extracted using a Power Soil1 DNA Isolation Kit (MoBio) according to the manufacturer’s instructions. PCR amplification and pyrosequencing DNA fragments of approximately 500 bp of the bacterial 16S rRNA gene, targeting the hypervariable region V1–V3, were amplified using the primer pair 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 533R (50 -TTACCGCGGCTGCTGGCAC-30 ) fused with the 454 Life Sciences primers, barcodes and adapters. The PCR amplifications were carried out in triplicate 50-mL reactions containing 10 ng metagenomic DNA, 2.5U Platinum Taq DNA Polymerase (Invitrogen), 1 mM of each barcoded fusion primer, 0.2 mM of dNTPs in the appropriate 10 PCR Buffer and de-ionized ultrapure water. Negative control samples were treated similarly with the exclusion of template DNA. PCR reactions were started by an initial denaturation at 94 °C for 30 s, followed by five cycles of 94 °C for 20 s, 45 °C for 20 s, 65 °C for 60 s, followed by 20 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s, with a final extension step of 5 min at 72 °C. The PCR products from replicated reactions of the same sample were pooled and purified from 1.5% agarose gels with a QIAquick Gel Extraction Kit (Qiagen). Purified DNA was quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). DNA amplicons were then combined in equimolar ratios into a single tube. Pyrosequencing was performed using the 454 GS FLX platform (Roche). Data processing and bacterial population analysis Data were processed by following the pipelines with MOTHUR [31]. Sequences with ambiguous bases, primer mismatches, homopolymer runs in excess of six bases or errors in barcodes, and with length shorter than 200 bp, or average quality score
