Characterization and Identification of Neocosmospora solani and Fusarium oxysporum Causing Root Necrosis and Wilting of Orange Trees in Chile

Abstract

Orange trees (Citrus × sinensis (L.) Osbeck) are the third-most cultivated citrus fruit species in Chile. In recent years, several trees in three orange orchards of 'Lane late' and 'Fukumoto' cultivars grafted on 'Robidoux' trifoliate orange (Poncirus trifoliata (L.) Raf.) have shown chlorosis, canopy reduction, wilting, root necrosis, defoliation, and plant death symptoms. This study aims to characterize the morphological symptoms observed in diseased orange trees in central Chile and identify the fungal pathogens that are involved. Isolation and morphological characterization of the pathogens were conducted by using different culture media. A total of 53 isolates were obtained, morphologically characterized and 12 isolates were selected for molecular identification. The isolates were identified using ITS, TEF-1α, and RPB2 regions. Two Fusarium species complexes were identified, Neocosmospora (Fusarium) solani (FSSC) and F. oxysporum (FOSC), based on >99% identity. Pathogenicity tests were conducted on young orange seedlings under greenhouse conditions. Results indicated that two months post inoculation, trifoliate orange seedlings displayed root rot symptoms such as necrosis, vascular discoloration, and wilting. FSSC and FOSC were re-isolated from necrotic seedling roots and identified through a combination of morphological traits and molecular techniques. This is the first detailed report of this disease, attributed to FSSC and FOSC, in orange orchards in Chile. These diagnostic results represent the first step in developing adequate phytosanitary programs for managing this disease.

Keywords: Fusarium spp.; citrus; dry root rot; morphology; phylogenetic analysis; vascular wilt.

Figure 1

Symptomatology of orange trees in orchards in Melipilla, Metropolitan Region, Chile. (A,B) Sparse canopy and yellowing; (C) partial defoliation; (D) fruit persistence in the canopy with wilting leaves and advanced decline stage; (E) chlorosis and epinasty of citrus leaves; (F,G) root rot and vascular wilt.

Figure 2

Morphological structures of Neocosmospora (Fusarium) solani isolated from orange trees. (A,B) Colonies on PDA after 7 days at 20 °C (view from upper site (A) and down site (B) of the petri dish); (C) aerial conidiophores formed on the surface of carnation leaves; (D–F) areal microconidia organized on mucilaginous false heads; (G,H) macro and microconidia; (I) Chlamydospores.

Figure 3

Morphological structures observed in F. oxysporum isolated from orange trees. (A,B) Colonies on PDA after 7 days at 20 °C (view from upper site (A) and down site (B) of the petri dish); (C) aerial conidiophores formed on the surface of carnation leaves; (D–F) areal microconidia organized on mucilaginous false heads; (G,H) macro and microconidia; (I) Chlamydospores.

Figure 4

Phylogenetic tree construction using the Maximum Parsimony method based on a concatenated dataset of TEF-1α, ITS, and RPB2 sequences of 47 strains belonging to the Fusarium species complex using MEGA v11. Support for the branches was evaluated using bootstrap analysis of 500 replications. Isolates obtained from orange trees are indicated in bold. The branch lengths were proportional to the distance. Fusicolla aquaeductuum (NRRL 20686) was used as an outgroup.

Figure 5

Necrosis generated by Fusarium spp. infection in three-month-old Rubidoux rootstock orange seedlings recovered 60 days post-inoculation. Uninoculated control (A), Neocosmospora (Fusarium) solani (B) and F. oxysporum (C).