Development of PCR-Based Race-Specific Markers for Differentiation of Indian Fusarium oxysporum f. sp. cubense, the Causal Agent of Fusarium Wilt in Banana

Abstract

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL−1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.

Diagnostics of Fusarium wilt in banana: Current status and challenges

“…3A,B) in Thangavelu et al. (2022). The lowest concentrations that generated successful amplification for Foc R1 and R4 were 1.0 and 0.1 pg/μL, respectively.…”

“…In addition, there are discrepancies between the described results for sensitivity and the referenced figures (fig. 3A,B) in Thangavelu et al (2022). The lowest concentrations that generated successful amplification for Foc R1 and R4…”

“…(2022) compared whole‐genome sequences of 61 isolates representative of Foc STR4, TR4 and R1, along with Foc reference genomes, and F. oxysporum f. sp. lycopersici isolates to identify unique regions in SIX gene sequences within races (Raman et al., 2021; Thangavelu et al., 2022). In this study TR4 isolates composed of VCG 01213/16 while all STR4 isolates belonged to VCG 0120.…”

“…Previously, the SIX8b gene allele was only found in STR4 isolates and not in TR4 (Carvalhais et al., 2019; Fraser‐Smith et al., 2014), but in isolates from India this allele was found in both STR4 and TR4 by testing 18 STR4 and TR4 isolates. To resolve this, the SIX7a gene allele was targeted for the detection of STR4 (Thangavelu et al., 2022). Targeting SIX7a can be problematic as it is also present in VCG 0126, which can be classified as R1, and VCG 01219, which is currently of an undetermined race (Czislowski et al., 2018).…”

Development and application of a droplet digital PCR assay for the detection of Fusarium oxysporum f. sp. cubense tropical race 4 from different biological samples

“…An important number of molecular markers for Foc TR4 detection have been developed to date (Aguayo et al., 2017; Carvalhais et al., 2019; Dita et al., 2010; Li, Zuo, et al., 2013; Lin et al., 2009, 2013; Ordóñez et al., 2019; Thangavelu et al., 2022).…”