Xanthomonas citri subsp. citri citrus canker in São Paulo. Molecular epidemiology and biocontrol potential of bacteriophage and their depolymerases.

São Paulo state is by far the largest sweet orange producer in the world and the biggest exporter to the EU and USA. The 2016 global juice concentrate market alone was valued at US$ 89.56bn and is forecast to rise to US$ 117.89bn by 2025. This crucially important industry employs approximately 200 000 people and therefore has enormously positive social and economic impacts on São Paulo state and the country of Brazil.

The orange production industry is threatened by Asiatic Citrus Canker (ACC), a devastating and untreatable disease of citrus plants that is only preventable by the costly removal and destruction of infected trees and frequent application of environmentally damaging copper biocides to which resistance has emerged and is spreading. An ACC eradication policy, in place until 2009, has been replaced with a risk mitigation system that allows for differing levels of disease incidence in orchards depending on the fruits final usage. This has led to endemic disease throughout the state and increasing use of environmentally toxic copper biocides that threaten soil and water course biodiversity.

ACC is caused by bacterial infection Xanthomonas. citri subsp. citri (X. citri), which results in premature fruit drop, stem dieback and defoliation. We currently know little about the diversity of strains of bacteria causing ACC in São Paulo as there have been few published studies and most of these have used poorly discriminatory methods that are unsuitable for robust epidemiological or evolutionary analyses. Genomic DNA sequencing is the gold-standard method for examining bacterial pathogens and tracing transmission and evolution of antimicrobial resistance and virulence but this has only been applied in a small number of published studies of X. citri that include just one isolate from São Paulo.

In this project we will examine the genetic diversity and epidemiology of X. citri from infected plants in Sao Paulo using genome sequencing. We will sample plants from 48 sites throughout the state in the summer of 2018/19 when disease incidence is highest and compare these to historical isolates and to those from other countries. This study of X. citri sampled from regions with high, medium and low levels of ACC will yield important information on how ACC has spread, whether hyper-virulent strains are present, frequency of DNA exchange with strains of the same and other species, and the scale and nature of the problem of copper resistance.

We will also isolate and characterize X. citri - specific viruses known as phage. These are proposed as an alternative to antibiotics in human health and they are currently used in the food industry e.g. for controlling Listeria contamination in cheese production. They are also used in the control of infections in tomato and pepper crops in the USA. We will assemble a collection of ~100 phage that we will test for their ability to kill X. citri isolates in laboratory studies and also their ability to protect from ACC in greenhouse studies. Through phage genome sequencing we have identified genes that produce enzymes with the potential to disrupt X. citri communities on leaves that exist as biofilms. These biofilms are held together by a polysaccharide 'slime' produced by the bacteria that helps it survive in the environment and is a key factor in causing disease. We will identify and produce a repertoire of these phage polysaccharide depolymerase enzymes and test their anti-biofilm activity in the laboratory and in greenhouse studies. We will also produce modified depolymerases with leaf-binding motifs that may promote their effectiveness in the field.

This study will give important new information on ACC disease in São Paulo to guide future crop-management and disease prevention strategies. It will also produce potential biodegradable and environmentally sustainable alternatives to copper biocides for possible field trials in 2021.

Who will benefit from this research and how will this happen?

Asiatic Citrus Canker (ACC) is a severe and untreatable disease of citrus trees that represents a major on-going threat to sweet orange production in São Paulo - an industry that employs around 200 000 people in the state and is worth in excess of US$32 bn annually. In this study we will examine the epidemiology of this endemic disease by studying genomic and population diversity within the bacteria that causes this disease - Xanthomonas citri subsp. citri (X. citri). This will help us track the spread of the disease, examine whether particularly aggressive types of the bacteria are circulating and determine the level and genetic basis of resistance and tolerance to copper biocides. These environmentally damaging biocides represent a severe threat to soil and especially aquatic biodiversity. They require frequent application and their action is preventative - they have no curative potential. Use of these heavy metal based biocides has increased since a state-wide eradication policy was ended in 2009 and new risk-mitigation strategies employed instead.

Knowledge of the diversity of the pathogen population across São Paulo will help us select lytic bacteriophages for potential use as species-specific, environmentally sustainable biocontrol agents to target ACC infections. We will also investigate the potential of using phage borne polysaccharides for infection prevention. Both approaches offer environmentally friendly and sustainable approaches to ACC control.

People whose livelihoods depend on the sweet orange production industry in São Paulo will be the primary beneficiaries of this research but it will also greatly help members of Dr Ferreira's and Prof Enright's research groups, particularly those directly engaged with the project. This award will also benefit scientists interested in plant-microbe interactions, microbial genomics and bacteriophage biology through the timely dissemination of large amounts of novel genomic sequences from bacterial and lytic bacteriophage sequencing and associated metadata. Our research may have important medium to long-term benefits in reducing the amounts of heavy metal pollution entering the ecosystem around sweet orange orchards and consequent increases in biodiversity in surrounding soils and watercourses. It may also serve to reduce the positive selective pressure for AMR in this economically key industry and with associated wider societal benefits.