Characterization of pathogens causing canker and dieback diseases in moringa (Moringa oleifera Lam.)

Abstract

Canker and dieback are increasingly recognized as major constraints in moringa cultivation, despite the crop’s well-known resilience and rich phytochemical defence. Field observations across Kerala have indicated a rising incidence of these diseases affecting young and mature plants alike, yet systematic documentation of the causal pathogens and their interaction with the host remains limited. The present investigation was undertaken to identify, characterize and evaluate the pathogenic potential of fungi associated with canker and dieback diseases of moringa, and further to assess host biochemical responses at critical developmental stages. The study was carried out in the Department of Plant Pathology, College of Agriculture, Vellanikkara, from 2023 to 2025. A purposive survey of moringa-growing homesteads and nurseries in Thrissur (AEU 10) and Ernakulam (AEU 12) districts yielded a total of 65 diseased samples from which 77 fungal isolates were obtained, comprising 50 from canker-affected tissues and 27 from dieback samples. Disease intensity varied among locations, with the highest PD1 for canker recorded at Kolazhy (AEU 10) (89.28 %), while the maximum PD1 for dieback was recorded at Kothamangalam (AEU 12) (76 %). Several samples yielded multiple isolates, indicating the coexistence of distinct fungal species within the same lesion. Isolates were purified using hyphal-tip culture and maintained on PDA for further studies. Canker symptoms under natural conditions included superficial bark cracking, pale-yellow to light-brown sunken or flat lesions and localized necrosis. Dieback symptoms, on the other hand, were characterized by progressive twig/branch drying, extensive cortical discoloration and the appearance of black, pinhead-like pycnidial structures on severely affected tissues. Pathogenicity testing conducted under rain-shelter conditions confirmed the ability of all tested isolates to reproduce disease symptoms on moringa, fulfilling Koch’s postulates. Symptom development varied markedly among isolates, indicating a wide spectrum of virulence. Cultural and morphological characterization of the isolates revealed considerable diversity in colony growth, pigmentation, sporulation and microscopic traits. Isolates associated with canker generally produced fast-spreading, cottony to fluffy mycelium with pink, white, or grey colouration, while dieback-associated isolates commonly showed dark, melanised mycelium with abundant pycnidial formation. Molecular characterization using ITS, TEF-1α and RPB2 gene regions confirmed the presence of multiple pathogenic species on moringa. Canker symptoms were associated with Fusarium irregulare, F. citri, Nectria sp. and Lasiodiplodia theobromae, whereas dieback was predominantly caused by L. theobromae, Curvularia sp. and Diaporthe batatas. This represents the first comprehensive documentation of the canker-dieback pathogen complex occurring on moringa in Kerala. Extracellular cellulases and pectinases production was evaluated using substrate-amended media. All isolates exhibited measurable cellulolytic and pectinolytic activity. Among the 77 tested isolates, several, including Fusarium sp. (AIS5), Fusarium sp. (VLD2), Lasiodiplodia sp. (VLD1), Fusarium sp. (MAD), Curvularia sp. (MLD2) and Curvularia sp. (AVD3), produced large hydrolytic zones (mm) within just 2-3 days, reflecting a strong enzymatic activity. Statistical analysis 2 revealed a highly significant positive correlation between lesion size and enzyme activity, indicating that both cellulase and pectinase secretion are strongly linked to virulence expression. Two highly virulent isolates, L. theobromae, CHS2 (canker) and L. theobromae, TRD1 (dieback), were selected for biochemical assays. Host response was examined at five principal developmental stages: leaf, shoot, flowering, fruiting and senescence. CHS2 produced small, slowly enlarging lesions across all developmental stages, beginning as light-brown or pale-yellow spots (0.1-0.2 cm) that expanded slowly and developed into ovoid to spindle-shaped forms with mild cracking by day five. In contrast, TRD1 generated faster-expanding lesions even in early stages, initial pale brown patches (0.1-0.2 cm) rapidly enlarged (up to 2.5 cm by day 5) and frequently coalesced, progressing from pale yellow or water-soaked patches to darker brown or black lesions. Overall, CHS2 showed slower, stage-dependent lesion expansion, whereas TRD1 exhibited rapid, aggressive lesion development across all growth stages. Total phenolic content varied widely among growth stages and according to pathogens. In the flower development stage, inoculated plants accumulated substantially higher phenols than controls from one day after pathogen inoculation, indicating a pronounced early defence response. Conversely, in leaf and fruit development stages, phenol levels in inoculated plants dropped significantly below those of the corresponding controls, suggesting growth stage-specific phenol biosynthesis in response to pathogen infection. During senescence, phenol levels remained low across TRD1 inoculated treatment, reflecting a natural decline in defence capacity. Flavonoid content showed a transient decline at one to two days post inoculation across most developmental stages. This was followed by a clear rebound on days three and five, especially in the leaf and shoot development stages, reflecting induced defence activation. In the leaf development stage inoculated with CHS2, flavonoids increased markedly by day five (22.85 mg QE g⁻¹; ~26% above control). TRD1-inoculated tissues displayed a similar pattern of early reduction followed by strong accumulation of flavonoids from day three. Overall, the study establishes that canker and dieback in moringa are caused by a diverse assemblage of fungal pathogens exhibiting wide variability in virulence and extracellular enzyme activity. The findings highlight the central role of cell-wall degrading enzymes in facilitating tissue colonization and disease progression. Moreover, phenolic and flavonoid profiling across growth stages reveals that moringa exhibits a highly stage-dependent defence strategy, with stronger biochemical activation during early vegetative growth and flowering stages and weaker responses during fruiting and senescence. These insights form an essential foundation for developing stage-specific disease management strategies and for future exploration of host resistance mechanisms in moringa.