Molecular diagnostics of Paecilomyces formosus in Zagros forests using species-specific primers

Document Type : Research Paper

Authors

Department of Plant Protection, College of Agriculture, Razi University, Kermanshah, Iran

Abstract

Accurate identification of the host species is critically important for disease detection and informing appropriate disease management decisions. Paecilomyces formosus, a causal agent of dieback and decline of oak, is an emerging threat that may cause severe risk to the Zagros forests of Iran in the future. In this study, a nested PCR assay for the identification of P. formosus was developed with the species-specific primer pairs PaMF and PaMR designed from the comparisons of nucleotide sequences of the nuclear ribosomal DNA internal transcribed space regions (ITS) from P. formosus isolates and other closely related taxa. To accomplish this, we sampled forest trees with dieback symptoms in Kermanshah and Ilam provinces. The Paecilomyces isolates were identified based on morphological characteristics, acid production on keratin sucrose agar medium, and sequencing of the ITS-rDNA region and part of the beta tubulin gene. Nested PCR was successfully amplified a 441 bp product exclusively from P. formosus genomic DNA, and no cross-reactions were observed with any other species, and also P. variotii. The nested PCR method can detect 100 fg of P. formosus genomic DNA. Sixty of symptomatic forest trees from seven locations in Zagros foreste were assayed, resulting in the discovery of Amygdalus lycioides, Cerasus avium, Cerasus microcarpa, Quercus libani, Acer spp., Acer monspessulanum, Ficus carica, Ziziphus spina-christi, Tamarix ramosissima and Ziziphus spina-christi as new host species, and all seven infested areas. To fulfill Koch’s postulates, the experiments were carried out on detached branches and attached healthy branches of trees at the forests in Kermanshah and Ilam provinces, Iran. The PCR-based method developed here can be used for a fast and reliable diagnosis of P. formosus, monitoring the epidemics, and assessing management strategies in Zagros forests.

Keywords

Extended Abstract

Introduction

   Accurate identification of the host species is critically important for disease detection and informing appropriate disease management decisions. Paecilomyces formosus, a causal agent of dieback and decline of oak, is an emerging threat that may cause severe risk to the Zagros forests of Iran in the future. The present study was aimed to develop a nested PCR assay for the identification of P. formosus with the species-specific primer pairs designed from the comparisons of nucleotide sequences of the nuclear ribosomal DNA internal transcribed space regions (ITS) from P. formosus isolates and other closely related taxa.

 

Materials and Methods

    During 2020 to 2021, forest trees that exhibit branch dieback symptoms were sampled for fungal isolations from Ilam and Kermanshah provinces. The causal agent was isolated and purified using routine plant pathology methods. Paecilomyces formosus isolates were identified using a combination of morphological and sequence data from ITS-rDNA region and beta tubulin gene. Seqeunce data of ITS-rDNA region for all of   Paecilomyces were obtained from GenBank and aligned together with seqeunce data gnerated in this study. A pair of species-specific primer (PaMF and PaMR) with expected amplicon size of 441 bp was designed for P. formosus. Efficacy of this primer set was tested and verified on DNA extracted from P. formosus and other fungal species isolated from forest trees. To fulfill Koch’s postulates, the experiments were carried out on detached branches and attached healthy branches of trees at the forests in Kermanshah and Ilam provinces, Iran.

 

Results and Discussion

    Colony culture, micro-morphological and physiological characteristics of the fungal isolates recoverd from forest trees with dieback symptoms matched description of P. formosus. Blast search analysis of the ITS, and β-tub sequence data obtained in this study against the sequence data in GenBank confirmed the identity of the isolates as P. formosus. The result of the multigene phylogenetic analysis was in accordance with the molecular identification based on DNA sequences in BLAST search, thus resolving the morphological identification. Nested PCR was successfully amplified a 441 bp product exclusively from P. formosus genomic DNA, and no cross-reactions were observed with any other species. The nested PCR method can detect 100 fg of P. formosus genomic DNA. Sixty of symptomatic forest trees from seven locations in Zagros foreste were assayed, resulting in the discovery of Amygdalus lycioides, Cerasus avium, Cerasus microcarpa, Quercus libani, Acer spp., Acer monspessulanum, Ficus carica, Ziziphus spina-christi, Tamarix ramosissima, and Ziziphus spina-christi, as new host species, and all seven infested areas.

 

Conclusion

   The PCR-based method developed here can be used for a fast and reliable diagnosis of P. formosus, monitoring the epidemics, and assessing management strategies in Zagros forests.

References

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Iranian Journal of Plant Protection Science
Volume 54, Issue 1
September 2023
Pages 117-131

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History

  • Receive Date: 05 May 2023
  • Revise Date: 05 June 2023
  • Accept Date: 04 September 2023

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