Avilés-Noriega, Ashley

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    Sigatoka disease complex of Musa spp. in Puerto Rico and the development of a transformation vector for Pseudocercospora fijiensis to examine the avirulence 4 (PfAvr4) effector during infection of Musa spp.
    (2021-05-14) Avilés-Noriega, Ashley; Rivera-Vargas, Lydia I.; College of Agricultural Sciences; Giraldo-Zapata, Martha C.; Cuevas, Hugo E.; Department of Crops and Agro-Environmental Sciences; Cabrera-Ríos, Mauricio
    Bananas and plantains (Musa spp.) are among the world’s most economically important crops. Worldwide, the most important fungal leaf diseases of Musa spp. are the Sigatoka disease complex, comprised of the black leaf streak (Pseudocercospora fijiensis), the Sigatoka leaf spot (P. musae), and the Eumusae leaf spot (P. eumusae). This last species has not been reported in the Western Hemisphere. Considering the rapid rate of spread of black Sigatoka in Puerto Rico after its first observation in 2004, and the potential threat to Musa spp. by P. eumusae, the first objective of this study was to reassess the Sigatoka disease complex in the island. Sixty-one leaf samples were collected from thirteen municipalities around the island showing Sigatoka-like symptoms. Leaf samples were placed on petri-dish humid chambers for 24 h to establish monosporic cultures, colony morphology was recorded and described by stereoscopic and microscopic observations and measurements. Genomic DNA was extracted from leaf samples and fungal colonies. PCR amplifications were performed using universal and species-specific primers. To support fungal identification, amplicons were sequenced and analyzed by constructing Maximum- Likelihood (RAxML) phylogenetic trees. Fifty leaf samples amplified with P. fijiensisspecific primers, P. musae, and P. eumusae were not detected, suggesting that Black Sigatoka has displaced the Yellow Sigatoka disease in the island. Fungal isolates were identified as P. fijiensis (n=24), Cladosporium spp. and Cladosporium-like spp. (n=4), C. dominicanum (n=1), Neocordana musae (n=2), Zasmidium spp. (n=6), and Z. musigenum (n=1). Other fungi identified from the Musa spp. samples belong to the genus Zygophiala sp. and Deightoniella sp. but were not successfully isolated. The second objective of this study focuses on the virulence factors produced by P. fijiensis to colonize Musa spp. and understanding their plant-pathogen interactions which remain unclear. For this reason, we developed a transformation vector to study P. fijiensis using the green fluorescent protein (GFP) to examine potential virulence factors during the infection processes. We compared P. fijiensis infection process on leaf sheaths and undetached leaves. The leaf sheath inoculation method was the best approach to study P. fijiensis infection process, revealing pseudohyphal-like bulbous invasive hyphae that have never been described before for this pathogen. Designed primers successfully amplified the PfAvr4 gene of P. fijiensis. A transformation vector based on the Agrobacterium tumefaciens pBHt2 plasmid was developed to evaluate PfAvr4 expression with an enhanced GFP (EGFP). The leaf sheath inoculation method and transformation approaches employing the vector developed in this study could be used to elucidate effector interactions in the Sigatoka pathogens when colonizing Musa spp. Accurate identification of species causing leaf diseases in Musa spp. will allow establishing quarantine regulations and specific management approaches in Puerto Rico. Furthermore, understanding molecular plant-pathogen interactions of these very important fungal pathogens will contribute to the development of new management strategies.