Peeling the onion: Unravelling the biology of fungal onion pathogens causing leaf blight,neck rot and white rot

Research output: Thesisinternal PhD, WU

Abstract

Onion (Allium cepa) is a vegetable crop that is consumed and cultivated worldwide. The production of bulbs is threatened by pathogens and pests that infect onion plants and cause yield losses. Three severe diseases of onion are leaf blight, neck rot and white rot and all three are caused by fungi of the family Sclerotiniaceae. In chapter 1, the long history of Sclerotiniaceae diseases of onion is discussed, and an overview of the disease etiology and current control strategies is given. Leaf blight is a foliar disease of onion caused by Botrytis squamosa. Neck rot is a post-harvest disease that manifests in bulbs after storage and which can be caused by either of three Botrytis species: B. aclada, B. allii and B. byssoidea. White rot is a soil-borne disease of onion bulbs caused by Sclerotium cepivorum and is devastating to continued onion cultivation in production fields. In this thesis project, I studied several aspects of the plant-pathogen interaction with the aim to unravel the biology of Botrytis species and S. cepivorum pathogenic on onion. New insights into genetic, biochemical, molecular and cellular aspects of the interaction of Botrytis species and S. cepivorum with their host onion are essential to develop new breeding strategies for resistant onion cultivars as a durable solution to leaf blight, neck rot and white rot.

To better understand the infection biology of the fungal pathogens of onion, we transformed B. squamosa, B. aclada and S. cepivorum with the fluorescent label GFP that allowed to visualize the fungi in their first contact with host tissue in chapter 2. B. squamosa entered onion leaves by penetrating the leaf surface through stomata or by growth into anticlinal walls of onion epidermis cells. B. aclada did not penetrate the leaf surface but instead formed superficial colonies. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced these infection structures, but upon prolonged contact with the tomato root the structures died. Visualization of the infection of all three fungi helped to better understand the infection strategy of the pathogens.

Onion plants defend themselves against pathogens by producing compounds with antimicrobial activity. A previous study identified metabolites that belong to the class of saponins, called ceposides, that are present in onion and have antifungal activity. We tested the effect of ceposides on B. aclada and S. cepivorum and showed a growth inhibitory activity in a concentration-dependent manner in chapter 3. Since B. aclada and S. cepivorum are specialized pathogens of onion bulbs, we hypothesized that they likely evolved mechanism to tolerate ceposides. In the genomes of both species, we identified genes that may be involved in the detoxification of ceposides. These genes, encoding enzymes that can possibly hydrolyse the terminal sugar residues rhamnose and xylose, were tested for their involvement in ceposide detoxification.

In chapter 4, we attempted to pinpoint the genetic determinants of host specificity in the genus Botrytis. We sequenced the genomes of onion pathogenic Botrytis species and S. cepivorum, as well as genomes of Botrytis species pathogenic on other hosts. By comparing the genomes, we searched for genes underlying pathogenicity and host specificity for onion. The distribution of genes encoding secreted proteins and secondary metabolite gene clusters was analysed among all sequenced species. We were able to reconstruct the evolution of the genus Botrytis and found that chromosomal architecture had remained remarkably similar between species that diverged millions of years ago. In addition, evidence was provided for horizontal transfer of the gene cluster responsible for the production of the phytotoxic metabolite botcinic acid.

Necrotrophic pathogens such as B. squamosa use effector proteins that actively induce plant cell death by co-opting the programmed cell death machinery of the host. In chapter 5, we investigated the cell death-inducing activity of effector proteins of B. squamosa. In addition to studying effectors identified by comparative genomics, we assessed the protein composition of a B. squamosa culture filtrate that caused necrosis upon infiltration in onion leaves. The cell death-inducing activity of effector proteins was tested by transient expression via Agrobacterium tumefaciens-mediated transformation and by infiltrating proteins produced by Pichia pastoris. Besides the ability of effectors to induce cell death, we assessed their role in virulence and genetic diversity. One effector, Nep1, could rapidly induce a severe cell death response upon infiltration in onion.

The effector Nep1 is not unique for B. squamosa, since many Nep1-like proteins (NLPs) have been identified in other plant pathogenic microbes. NLPs were reported to have cytolytic activity exclusively on dicot plant species. Since we observed cell death in the monocot plant onion upon infiltration of B. squamosa Nep1, we studied the cytolytic activity of NLPs on monocots in detail in chapter 6. Eight onion genotypes appeared to be differentially sensitive to NLPs. Analysis of the GIPC sphingolipid composition, a trait that allegedly determines the hitherto assumed dicot-specific NLP activity, revealed that GIPC composition of onion genotypes did not correlate with their NLP sensitivity. By infiltrating Nep1 protein in an interspecific Allium hybrid population, we identified a QTL for BsNep1 insensitivity that co-localized with a previously identified QTL for B. squamosa resistance.

In chapter 7, aspects of the interaction of Botrytis species and S. cepivorum with their host onion are discussed. By integrating the genetic, biochemical, molecular and cellular insights, I describe our attempts to decipher what makes certain Botrytis species host-specific pathogens of onion. Furthermore, I discuss how the different aspects of pathogenicity on onion studied in this thesis can help to identify sources of resistance. Lastly, I describe my vision about the consequences of the differences in pathogen biology for developing resistance breeding strategies as a durable solution against leaf blight, neck rot and white rot.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • van Kan, Jan, Promotor
  • Scholten, Olga, Co-promotor
Award date28 Jun 2021
Place of PublicationWageningen
Publisher
Print ISBNs9789463958189
DOIs
Publication statusPublished - 2021

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