Diversity of sclerotium rolfsii infecting yams

Abstract

The study entitled “Diversity of Sclerotium rolfsii infecting yams” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2023-2025 with the objectives of cultural, morphological, and molecular characterization; assessment of cross infectivity and genetic variability of Sclerotium rolfsii infecting yams. A survey was conducted in six Agro-Ecological Units (AEUs) of Kerala comprising of districts viz.,Thiruvananthapuram (Vellanad, Aruvikkara, Nedumangad and Neyyatinkkara), Kollam (Kochanjilmoodu and Kottarakara), Alappuzha (Tamarakulam and Cherthala), Kozhikode (Orkkateri, Vadakara, Aroor, Chekkiad, Vattoli and Vattoli North) and Kannur (Chonadam and Thalassery). A total 32 diseased samples were collected from different yams viz Elephant foot yam (EFY), Colocasia, and Dioscorea from 16 distinct locations of the surveyed areas. Of the 32 isolates obtained 25 isolates were collected from EFY, four isolates from Colocasia, and three isolates from Dioscorea. Disease incidence (DI) in the surveyed areas showed a range between10.0% to 60.0%. The pathogenicity tests confirmed virulence of the pathogen, with symptoms manifesting between 3 to 6 days post- inoculation. EFY plants inoculated with the isolates toppled within 12-20 days, whereas Colocasia and Dioscorea plants failed to topple. Isolates IA17 (EFY) collected from Orkatteri (AEU10), IC4 (Colocasia) collected from Chonadam (AEU2) and ID3 (Dioscorea) collected from Cherthala (AEU1), were identified as the most virulent isolates, inducing symptoms within 3-4 days on their original host. Cultural and morphological characterization revealed significant diversity. Mycelial growth rate varied from 1.53 cm/day to 2.46 cm/day. The isolate IA13 with growth rate of 2.46cm/day was identified as fast grower. The fungal isolates exhibited rapid mycelial growth, completing full growth in petri plate(9cm) in 3-5 days, displaying colors ranging from white, pure white, to dull white without any pigmentation. Colony texture varied, from fan-like to thick-centered (IA3, IA7), fluffy (IA8, IA14), and compact (ID2), with regular margins (except IA3, IA7). Days for sclerotial initiation was between 7-14 days while isolates IA15 and ID2 did not produce sclerotia. Sclerotia were smooth, round, light cream to light/dark brown. Meanwhile after two weeks of sclerotial initiation the number of sclerotia ranged between 25 (IC1) to 211 (IA10). The weight of 100 sclerotia varied from 56mg (IC1) to 195mg (IA3), and their size from 0.90 mm (IA12) to 1.99 mm (IC2). Microscopic analysis confirmed the presence of clamp connections, with septal distance ranging from 20.90μm (IA20) to 136.29μm (IA7), and hyphal width from 1.30μm (IA18) to 6.23μm (IA7). The most virulent isolates (IA17, IC1, and ID2) were identified as Agroathelia/Athelia rolfsii (Teleomorph of S. rolfsii) through molecular characterization, using ITS primers and the sequences were submitted to GenBank. Cross-infectivity studies confirmed that all 32 isolates were capable of infecting all three yam host. The symptom development was delayed in cross- inoculated hosts (7–11 days) compared to the original host (3–6 days). Despite the absence of plant toppling symptoms, Colocasia and Dioscorea plants cross-inoculated with isolates (IA1 to IA25) showed more sclerotia with sparse mycelial growth, when compared to the symptoms of original host infection. Lesion sizes ranged from 10 cm2(ID1 x A) to 27.99 cm2 (IC4 x A) on Elephant Foot Yam (EFY), 8 cm2 (IA18 x C) to 32 cm2 (IA22 x C) on Colocasia, and 8 cm2 (IA8 x D) to a maximum of 42 cm2 (IA17 x D) on Dioscorea plants. Genetic diversity analysis using five SSR primers generated 182 scorable bands with polymorphism indicated substantial genotypic heterogeneity. Primers MB-14 and BJ112 exhibited the highest Polymorphism Information Content (PIC value of 0.5). Jaccard's similarity coefficient ranged from 0.00 to 1.00. Critically, pairs of isolates were identified as clones (e.g., IA18/IA19, IC2/IC4) with similarity index of 1.00. At a Jaccard similarity coefficient (JC) of 0.82, the fungal isolates resolved into three major phylogenetic clusters. One distinct cluster was formed by the isolates IA20 (AEU9) and IA21 (AEU9), indicating a high degree of genetic similarity between these two specific isolates. The 16 isolates of EFY formed the second, separate, and larger cluster. The remaining EFY isolates formed a third cluster with colcoasia and dioscorea isolates. Cluster analysis of this cluster indicated significant genotypic overlap between isolates from Elephant foot yam, Colocasia, and Dioscorea, suggesting successful, shared genotypes across different hosts, alongside the presence of highly dissimilar specialized genotypes. This comprehensive analysis confirms genetic diversity in the pathogenic S. rolfsii population infecting yams across Kerala.

Virulence assessment were confirmed by the detection of oxalic acid in the most virulent (IA17, IC4, and ID3) and lesser virulent (IA7, IC1, and ID1) isolates from High-Performance Liquid Chromatography (HPLC) analysis. The study confirmed that oxalic acid (Retention Time 3.2 min), is a key necrotrophic effector, in the culture filtrates of the S. rolfsii (7DAI) isolates from EFY, Colacasia and Dioscorea. Analysis of peak areas revealed a direct correlation between fungal virulence and the production of Oxalic acid. IA17, IC4 and ID3 were identified as the most virulent ones in pathogenicity assays as these isolates exhibited the highest oxalic acid content. Conversely, a lower content of oxalic acid was observed in the IA7, IC1, and ID1 isolates with less virulence. The present study demonstrated that S. rolfsii infecting yam species in Kerala is characterized by high levels of pathogenic, morphological, and genetic diversity.The cross infectivity studies showed polyphagous nature of S. rolfsii affecting yams. The isolates from different yams were clustered into three different clusters indicating variability. The demonstration of broad cross-infectivity and the detection of shared, highly successful genotypes across different hosts necessitates the implementation of integrated disease management strategies