Occurrence and infective potential of Colletotrichum gloeosporioides isolates associated to Citrus limon var Eureka

Abstract

A collection of 37 fungi associated to Italian lemon plants with disease symptoms, was obtained. Ten genera including Aspergillus, Alternaria, Nigrospora, Lasiodiplodia, Dothideomycetes, Pleurostoma, Setosphaeria, Penicillium, Fusarium and Colletotrichum were identified by using ITS1-5.8S-ITS2, D1/D2 26S and COX1 loci. The last three genera were abundant on the damaged fruits, being Colletotrichum the more abundant (32.4 %). CaInt2 and CgInt primers support the identity of these isolates as C. gloeosporioides. Variability, inferred by rep-PCR and multilocus sequence analysis shows genetic differences among the C. gloeosporioides isolates. Infective profile evaluated in Colletotrichum isolates shows different leave infection percentages (26 to 60 %). SEM analysis showed mycelium, spores and appressoria on the leaves of selected Colletotrichum isolates. Specifically, the AL-05 and AL-13 isolates showed a high chitin deacetylase activity (CDA) peaking at 1.2 U/mg protein in AL-13. This is the first report on C. gloeosporioides infecting Italian lemon leaves in Mexico.

Keywords: CDA activity; Colletotrichum; Italian lemon; Pathogenic potential.

Fig. 1

Phylogenetic dendrogram based on ITS1‐5.8S‐ITS2 regions from the fungal community of Citrus limon var. Eureka. The dendrogram was built using a maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap to infer topology.

Fig. 2

Phylogenetic dendrogram based on D1/D2 LSU regions from the fungal community of Citrus limon var. Eureka. The dendrogram was built using maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap to infer topology.

Fig. 3

Phylogenetic dendrogram based on COX1 gene from the fungal community of Citrus limon var. Eureka. The dendrogram was built using a maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap to infer topology.

Fig. 4

Genetic variability of the selected Colletotrichum isolates in the study inferred by rep-PCR analysis a) Left; UPGMA tree from absence/presence band information, right; PCR lanes organized according to UPGMA dendrogram. C. gloeosporioides (AL-05, AL-06, AL-08, AL-09, AL-11, AL-12, AL-13) from Italian lemon. C. gloeosporioides (Cg-06) and C. acutatum (Ca-93 and Ca-96) from avocado. b) Multilocus sequence analysis (D1/D2 26S, ITS1–5.8S–ITS2 and COX1) in the C. gloeosporioides isolates from Italian lemon. Concatenated ITS1–5.8S–ITS2, D1/D2 26S and COX1 sequences of the reference C. acutatum strain KC05 were used as outgroup.

Fig. 5

Discrimination between C. gloeosporioides and C. acutatum isolates by using species-specific primers. a) 1.5% agarose gel showing the specific amplification of the C. gloeosporioides strains with the CgInt and ITS4 primers, and b) 1.5% agarose gel showing the specific amplification of the C. acutatum strains with the CaInt2 primers and ITS4.

Fig. 6

Infective potential of the C. gloeosporioides isolates and other fungal abundant genera on leaves of the Italian lemon (Citrus limon var. Eureka). a) AL-09, b) AL-08, c) AL-06, d) AL-12, e) AL-13, f) AL-05, g) AL-11, h) Cg- 06, i) Ca-93, j) Ca-96, k) AL-38, l) AL-21, m) AL-20, and n) Negative control.

Fig. 7

SEM images of wounded leaves of Citrus limon var. Eureka with representative C. gloeosporioides isolates showing the mycelial growth at 6 days, and spores and appressoria structures. a) AL-06, b) AL-13, c) AL-05, and d) details of AL-13.

Fig. 8

Determination of the CDA of representative C. gloeosporioides; AL-13 (a) and AL-05 (b).