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Infectivity Assays and Sequence Analyses for Unassigned Pseudomonas Species as Putative Cause of Dieback Disease of Dalbergia Sissoo Roxb. in Bangladesh

Hanny Tantau
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Nayuf Valdez Aguirre
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Rakha Hari Sarker
Department of Botany, University of Dhaka, Dhaka

Jana Schulze
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Denise Palm
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Annika Stubbe
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

K. Shamimul Alam
Department of Botany, University of Dhaka, Dhaka

S, Mihir Lal Saha
Department of Botany, University of Dhaka, Dhaka

M. Salim Khan
BCSIR Laboratories, Tissue Culture Section, Dhaka

M. Imdadul Hoque
Department of Botany, University of Dhaka, Dhaka

Hans Peter Mühlbach
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Steffi Renk
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Dorothee Schultz
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Heidrun Meyer
Molecular Phytopathology and Genetics, Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

Abstract/ Executive Summary:

The causal agent(s) of dieback of sissoo (Dalbergia sissoo Roxb.) have not yet been identified unequivocally. Putative microbial pathogens (fungi and bacteria) were studied in dieback affected sissoo trees collected from Bangladesh, using plant pathological techniques combined with molecular tools. DNA based characterization showed the presence of heterogeneous patterns of various fungi (mostly saprophytic). It did not support the hypothesis of Fusarium solani being the cause of sissoo dieback. In contrast, isolation and molecular characterisation of bacteria from dieback affected sissoo revealed the presence of Pseudomonas in 83% of the samples. Sequencing the gene of 16S ribosomal RNA, the rpoD?gene, the gacAgene and thempB?gene strongly suggested that these isolates belong most probably to a still unassigned Pseudomonas species. Hypersensitivity response assays and infection studies using sissoo seedlings demonstrated their pathogenic potential.

Keywords: Sissoo, Dieback, Pseudomonas, Fungal pathogens

Location: Dhaka, Tangail, Sirajganj, Bogra, and along Jamuna Bridge Road, Rajshahi division

Start Date:

End Date:

Program Area: Pest Management

Commodity: Sisso

Objectives:

Fungi were identified via sequence analysis of the ribosomal internal transcribed spacer (ITS) DNA, while bacteria of the genus Pseudomonas were detected and characterized via sequencing of taxon specific genes and the following questions were addressed: (a) association of a pathogenic fungal species with dieback, (b) the number of cases where Pseudomonas was found in dieback affected sissoo, (c) molecular characterization of these Pseudomonas isolates, and (d) pathogenic potential of Pseudomonas isolates

Methodology:

Samples of leaves, twigs, barks and roots were collected from symptomless and from dieback affected Dalbergia sissoo Roxb. at various sites viz. Dhaka, Tangail, Sirajganj, Bogra, and along Jamuna Bridge Road, Rajshahi division, Bangladesh. Rating of severity of dieback symptoms was as follows: Class ‘0’: means no typical symptoms of dieback, ‘1’: mild symptoms such as leaf chlorosis and necrosis and initial crown transparency, ‘2’: medium symptoms such as strong leaf necrosis, advanced crown transparency and gummosis (black spots) at the bottom of the trunk, ‘3’: severely affected trees with almost all foliage and branches lost (staghead) and many black spots on the whole trunk. Collected samples were kept cool in sealed plastic bags during transport and finally stored at –70°C. For the detection of fungi, explants of sample materials (barks and roots) were surface disinfected and placed on three different agar media (Atlas and Snyder 2006, Pontecorvo et al. 1953 and Leach et al. 1982). Selected mycelia were subcultured until visual homogeneity and grown in submerse culture. DNA was extracted from the collected mycelia (Day and Shattock 1997) and analyzed in 0.8% agarose gels. The following primer pairs were used for PCR and for sequencing in order to identify the isolated fungi: ITS4 and ITS5 (White et al. 1990) for the amplification of the ITS?region of various fungi, and the taxon selective ITS?fu primers (Abd Elsalam et al. 2003) for the detection of Fusarium species. Sequences of both strands of PCR products were obtained using Big Dye chain terminating mixture (Applied Biosystems, Darmstadt, Germany) as per the manufacturer’s instructions, and used for database search (BLAST, NCBI). Bacterial colonies were isolated by homogenising 1 g of surface disinfected sample material in 50 ml of sterile 0.8% NaCl and plating six ten?fold dilutions on King’s B medium (King et al. 1954), GH medium and NS medium (Schaad 1980). Colony PCR and sequencing studies were done using the following primer pairs: 16S rDNA specific primers Y1 and Y2 (Young et al. 1991), and the taxon (Pseudomonas) selective primer pairs rpoD?f and rpoD?r (Mulet et al. 2009), gacA? 1F and gacA₂ (Costa et al. 2007) as well as rnpB?f (TAC GGA AAG TGC CAC AGA AAA) and rnpB?r (GGA GAG TCG ATC TRT AAG C) (Leif Kirsebom, personal communication) For transmission electron microscopy (TEM) of bacterial isolates, cells from overnight cultures were observed with TEM Leo 906 E (Carl Zeiss, Oberkochen, Germany) after negative staining with uranyl acetate. Scanning electron microscopy (SEM) of plant material was done using the Philips XL?20 scanning EM. Specimen preparation followed standard procedures using critical point drying and gold coating with sputter coater SCD 030 (Bal?Tec, Liechtenstein)

Source of Information: Plant Tissue Cult. & Biotech. 21(2): 101- 113, 2011 (December)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, present findings provide clear evidence in favour of the hypothesis that the bacteria Pseudomonas play an important role in the etiology of the dieback disease of Dalbergia sissoo, while various pathogenic and opportunistic fungi may be involved as secondary parasites. However, intensive future efforts are required for understanding and possibly controlling of this disastrous disease.

Knowledge Management: Journal