Identification of the causal agent of citrus bacterial canker and its biocontrol using epiphytic bacteria under greenhouse conditions

Abstract

Background and Objectives

Citrus is important for wealth generation and economic development in many countries worldwide. It is a subtropical fruit mainly originating in India, Iran, China, Japan, and some Southeast Asian countries. The bacterial canker is one of the most destructive citrus diseases that affects all citrus varieties and causes massive financial losses to the citrus industry worldwide, either in terms of a decrease in the quantity and quality of fruit production, reduced access to export markets, or the expenses associated with the prevention and control of the disease. Canker lesions begin as light yellow, raised, spongy blister-like formations on the leaves, twigs, and fruit surface. As the lesions enlarge, the spongy formations collapse, and brown depressions appear in their central portion, forming a crater-like appearance. This research aimed to identify the causal agent of citrus canker, isolate bacterial epiphytes from citrus trees (northern and southern regions), and examine their antagonistic activity against the pathogenic isolate under laboratory and greenhouse conditions.

Materials and Methods

In this study, epiphytic bacterial isolates from 100 samples of branch, leaf, and fruit of various citrus trees (orange, lemon, lime, etc.) obtained from Mazandaran, Gilan, Khuzestan, and Kerman provinces were isolated on a nutrient agar culture medium. Moreover, the pathogenic strain CC29 was isolated from infected Lime trees and identified based on phenotypic features, and a pathogenicity test was prepared by the Department of Plant Protection, Faculty of Agriculture of the University of Zanjan. The antagonistic activity of the epiphytic isolates against the pathogenic strain CC29 was measured by co-inoculation method under in vitro conditions. Antagonist isolates were selected and identified based on physiological and biochemical tests. In greenhouse experiments, the selected antagonistic epiphyte isolates were inoculated in the leaves of two-year-old seedlings of the Mexican lime cultivar two days before pathogen inoculation. The seedlings were kept in a greenhouse at 25◦C to 27◦C and 55 % to 60 % relative humidity. After one month, the disease severity was calculated using Motic Images Advanced 3.2 software. This experiment was conducted in a completely randomized statistical design in four repetitions. Data analysis was performed using SAS version 9.4 software.

Results

In this study, molecular identification based on the partial 16S rRNA gene nucleotide sequences, the pathogenic isolate showed the highest similarity to the type strain Pantoea agglomerans strain H68. In the antagonistic activity tests, Out of 311 isolates, 82 isolates were selected based on negative results in the hypersensitivity reaction on tobacco and geranium leaves and soft rot on potato slices. In in vitro conditions, 32 isolates showed an inhibition zone against Pantoea agglomerans strain CC29, with the largest inhibition zone diameter being five millimeters for isolate 45. In the greenhouse condition, the antagonistic activity of 13 selected epiphyte isolates against strain CC29 was investigated in the leaf tissue. The variance analysis of data showed that the effects of treatments on the reduction of symptom severity were significant at the 1 % level. The epiphytic isolate 22 reduced the disease severity compared to the positive control and had the greatest effect in reducing the spread of symptoms. These isolates belong to the genus Pseudomonas sp. based on phenotypic characteristics.

Discussion

This study demonstrated that Pantoa agglomerance has the potential to play diverse roles in interactions with plants, as previous studies have shown. The strains of this species have the potential to act as a causal agent of disease in one host and as a biological control agent in others. Because this species is used as a biological control agent for fungal and bacterial plant pathogens, it is essential to be aware of its pathogenic potential to prevent undesirable or dangerous effects. Although the antagonistic activity of bacterial isolates in vitro conditions is acceptable. However, the results may differ in vivo conditions due to the unpredictable behavior of antagonistic organisms in different conditions. According to the results, it would be beneficial to investigate the interaction between Pantoea agglomerans strain CC29 identified in this study and Xanthomonas citri subsp. citri.