High‐Throughput Assessment and Genetic Investigation of Vegetative Compatibility in Verticillium dahliae

Abstract

Classification of isolates into vegetative compatibility groups (VCGs) using nitrate‐non‐utilizing (nit) mutants has been widely used for the characterization of Verticillium dahliae populations. However, certain methodological limitations prevent its application on a large scale. Furthermore, systematic investigations into the genetics underlying complementation tests between nit mutants of fungal isolates (i.e. heterokaryon formation) are lacking for Verticillium species. In this work, a diverse collection of 27 V. dahliae isolates – including representatives of all VCGs, both mating types, and heterokaryon self‐incompatible isolates – was employed for the development and optimization of (i) a protocol for the rapid generation of nit mutants of V. dahliae isolates using UV‐irradiation and (ii) a reproducible high‐throughput procedure for complementation tests between nit mutants in liquid cultures using 96‐well microplates. The genetic analysis of selected heterokaryons demonstrated that the frequently encountered ‘weak’ cross‐reactions between VCGs and their subgroups can be actually heterokaryotic, implying the absence of strict genetic barriers between VCGs. In conclusion, we provide in this work an optimized method for the high‐throughput VCG assignment of V. dahliae populations and a genetic analysis of heterokaryons that may have serious implications for the interpretation of VCG classification data. These advancements in the available methodology and the genetic background of vegetative compatibility grouping may contribute to a better understanding of the population biology of V. dahliae and possibly other mitosporic fungi.

Hex1, the Major Component of Woronin Bodies, is Required for Normal Development, Pathogenicity and Stress Response in the Plant Pathogenic FungusVerticillium dahliae

“…Mutants of the Δhex1 strain were compatible with each other and gave rise to robust heterokaryons, suggesting that hex1 is not required for hyphal fusion. Mutants of both the wild-type isolate Ls.17 and its Δhex1 descendant exhibited strong reactions with members of the same compatibility group, as expected, and also weaker or inconsistent interactions with members of other groups, which is often observed in this species [40]. No difference in the compatibility profile of the Δhex1 mutant was recorded, suggesting that the gene is not involved in the genetic control of heterokaryon incompatibility.…”

An efficient method for selecting stable tester strains of vegetative compatibility groups in Verticillium dahliae

“…The Dutch testers of two main vegetative compatibility groups in V. dahliae did not make heterokaryons with themselves, as did American testers of these groups. This result confirms the conclusions of Papaioannou and Typas (2015) about the absence of strict genetic barriers between VCGs in V. dahliae. According to the authors a spectrum between highly stable and more unstable heterokaryosis exists in V. dahliae.…”

Two genomes are better than one: history, genetics, and biotechnological applications of fungal heterokaryons

“…Later researchers refined Puhalla’s techniques and identified four main nit phenotypes, which are referred to as nit 1, nit 2, nit 3, and nit M [ 51 , 52 ]. The most commonly reported phenotypes recovered are nit 1 and nit M [ 53 – 55 ], and pairing of these is used to visualize complementation and heterokaryon formation for a variety of applications. Due to its simplicity, the use of the nit mutant system has become a standard test for grouping fungal isolates into vegetative compatibility groups (VCGs), which consist of all strains of a fungal species that are capable of undergoing vegetative fusion and stable heterokaryon formation with each other [ 46 ].…”

“…Recently, a high-throughput method for obtaining nit mutants and performing complementation tests has been developed and tested [ 55 ]. This method speeds the generation of nit mutants by exposing them to ultraviolet radiation on chlorate-containing media.…”

“…Another potential pitfall of VCG analysis is the ability of members of different VCGs to form “weak” heterokaryons [ 81 ]. While often unstable and short-lived [ 55 ], these heterokaryons have shown that exchange of genetic material is possible between members of different VCGs.…”