pathogens Review The Destructive Fungal Pathogen Botrytis cinerea—Insights from Genes Studied with Mutant Analysis 1, 1,2, 1,2 1,2, Nicholas Cheung y , Lei Tian y, Xueru Liu and Xin Li * 1 Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; [email protected] (N.C.); [email protected] (L.T.); [email protected] (X.L.) 2 Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada * Correspondence: [email protected] These authors contributed equally to the review. y Received: 8 October 2020; Accepted: 4 November 2020; Published: 7 November 2020 Abstract: Botrytis cinerea is one of the most destructive fungal pathogens affecting numerous plant hosts, including many important crop species. As a molecularly under-studied organism, its genome was only sequenced at the beginning of this century and it was recently updated with improved gene annotation and completeness. In this review, we summarize key molecular studies on B. cinerea developmental and pathogenesis processes, specifically on genes studied comprehensively with mutant analysis. Analyses of these studies have unveiled key genes in the biological processes of this pathogen, including hyphal growth, sclerotial formation, conidiation, pathogenicity and melanization. In addition, our synthesis has uncovered gaps in the present knowledge regarding development and virulence mechanisms. We hope this review will serve to enhance the knowledge of the biological mechanisms behind this notorious fungal pathogen. Keywords: airborne fungal pathogen; Botrytis cinerea; fungal pathogenesis; sclerotial development; fungal growth; conidiation; melanization 1. Introduction Ascomycete Botrytis cinerea is a fungal pathogen responsible for gray (or grey) mold diseases. It has a broad host range, affecting many important agricultural crops. First described by Christiaan Hendrik Persoon in 1794 [1], it is considered a species within Botrytis, as it is the major pathogen within the genus [2]. Botryotinia spp. and Botrytis spp. previously caused taxonomy confusion because they induce similar disease symptoms as Sclerotinia spp. In 1949, Gregory confirmed that Botryotinia fuckeliana was the apothecial (sexual) stage of B. cinerea, establishing Botryotinia spp. and Botrytis spp. as anamorphs and teleomorphs of the same fungus [3]. B. cinerea belongs to the Sclerotiniaceae family of the class Leotiomycetes [4]. The Botrytis genus as to date comprises of 32 species [5]. Members of Botrytis genus are generally necrotrophic pathogens; they induce host cell death and lysis to access cellular nutrients. An exception to this is B. deweyae, which grows almost asymptomatically within Hemerocallis host species as an endophyte [6]. Botrytis species usually have a narrow host range, many are considered specialist pathogens that are restricted to infecting a single species or a small group of related species [7]. However, B. cinerea and B. pseudocinerea are generalists, with B. cinerea causing grey mold diseases in over 200 plants species [8]. Furthermore, B. cinerea is considered of higher agricultural and scientific importance due to its tendency of developing fungicide resistance [9]. B. cinerea is a highly successful pathogen due to its flexible infection modes, high reproductive output, wide host range and ability to survive for extended periods as conidia and/or small hardened Pathogens 2020, 9, 923; doi:10.3390/pathogens9110923 www.mdpi.com/journal/pathogens Pathogens 2020, 9, x B. cinerea is a highly successful pathogen due to its flexible infection modes, high reproductive output,Pathogens wide2020, 9,host 923 range and ability to survive for extended periods as conidia and/or small2 of 46 hardened mycelia masses called sclerotia [8]. It is primarily airborne as asexual conidia spores from maturemycelia conidiophores masses called serve sclerotia as major [8]. Itmeans is primarily of transmission airborne (Figure as asexual 1). Mycelia conidia sporesfrom sclerotia from mature and otherconidiophores infected tissues serve as as well major as meansseeds serve of transmission as alternative (Figure inoculum.1). Mycelia In addition from to sclerotia its asexual and cycle, other sclerotiainfected of tissues B. cinerea as well can as seedsundergo serve a asheterothallic alternative inoculum.sexual cycle In additionto form toapothecia, its asexual which cycle, release sclerotia ascosporesof B. cinerea [8,9].can Production undergo a heterothallic of ascospores sexual from cycleapothecia to form involves apothecia, the fertilization which release of ascosporessclerotia with [8,9 ]. microconidiaProduction of from ascospores an opposite from mating apothecia type. involves However, the fertilization the B. cinerea of sclerotiasexual cycle with is microconidia rarely observed from inan nature opposite [8]. mating type. However, the B. cinerea sexual cycle is rarely observed in nature [8]. InvasionInvasion of of host plantsplants by byB. B. cinerea cinereacan can start start from from damaged damaged tissues tissues or natural or natural openings, openings, allowing allowingthe fungus the tofungus establish to establish infection infection [10]. Initial [10]. infected Initial infected tissue often tissue results often in results limited in damage. limited damage. However, However,massive conidia massive production conidia production enables fast enables subsequent fast spreading.subsequent For spreading. fruiting plants, For fruiting the sites plants, of infection the sitesare usuallyof infection the floralare usually organs the and floral the fungusorgans hasand diverse the fungus means has of diverse infecting means different of infecting species. different In grapes, species.conidia In infect grapes, and spread conidia in theinfect receptacle and spread area [11 in]. Inthe raspberries receptacle and area strawberries, [11]. In theraspberries stigmatic and fluid strawberries,serves as adhesive the stigmatic and nutrient fluid mediumserves as for adhesive the airborne and nutrient conidia tomedium germinate for andthe airborne develop hyphaeconidia [to12 ]. germinateMycelia can and then develop grow hyphae along pathway[12]. Mycelia normally can then taken grow by along pollen pathway grains, thereby normally entering taken by the pollen carpel grains,and invading thereby theentering undeveloped the carpel ovaries. and invading the undeveloped ovaries. FigureFigure 1. 1. LifeLife cycle cycle of of BotrytisBotrytis cinerea. cinerea. After initial infection, B. cinerea enters a short phase where it exists as a biotroph within the After initial infection, B. cinerea enters a short phase where it exists as a biotroph within the plantplant [8]. [8]. Later Later on, on, it itenters enters an an aggressive aggressive necrotro necrotrophicphic phase, phase, which which is isprop proposedosed to to be be triggered triggered by by biochemicalbiochemical changes changes such such as as an an increase increase in in volatile volatile organic organic compounds, compounds, sugar sugar and and nitrogen nitrogen contents contents inin the the ripening ripening host host tissues tissues [13,14 [13,14].]. During During this this stage, stage, the the fungus fungus secretes secretes virulence virulence factors factors such such as as oxalicoxalic acid acid (OA), (OA), cell cell wall wall degrading degrading enzymes enzymes (C (CWDEs)WDEs) and and analogues analogues of of plant plant hormones hormones to to disrupt disrupt hosthost metabolism, metabolism, immune immune system system an andd cellular cellular structure structure [15,16]. [15,16 ].Effects Effects of of virulence virulence factors factors on on hosts hosts areare characterized characterized by by fruit fruit decay, resultingresulting inin softeningsoftening of of the the flesh flesh and and a browning,a browning, leathery leathery skin skin [17 ]. B. cinerea [17]. B. cinereaitself itself undergoes undergoes rapid rapid growth grow ofth mycelia of mycelia on plant on plant surfaces surfaces and forms and forms massive massive grey conidia grey conidiamasses masses [8]. At [8]. the At same the time,same adjacenttime, adjacent plant tissueplant tissue may become may become infected, infected, allowing allowing for the for spread the B. cinerea spreadof grey of mold grey acrossmold across the whole the whole plant andplant to and plants to plants nearby. nearby. Upon Upon killing killing the host the tissue,host tissue, B. cinereacan continue can continue to grow to saprophyticallygrow saprophytically within within the plant the remainsplant remains in the formin the of form sclerotia of sclerotia and mycelia. and B. cinerea mycelia.The sclerotia The sclerotia of of B.are cinerea highly are resistant highlyto resistant environmental to environmental changes. They changes. can remain They can viable remain in soil β viablefor up in to soil 360 days,for up likely to 360 due days, to their likely melanized due to surface, their melanized-glucan matrixsurface, and β-glucan intracellular matrix nutrient and intracellularreserves such nutrient as protein, reserves glycogen, such as polyphosphate protein, glycogen, and lipidspolyphosphat [18,19]. e and lipids [18,19]. HostsHosts of of B.B. cinerea cinerea includeinclude over over 500 speciesspecies ofof mostlymostly dicotyledons dicotyledons and and some some monocotyledons monocotyledons [8 ], [8],many many are ofare economic of economic importance. importance. Most severelyMost severely affected affected are agricultural are agricultural crops include