Emerging Approaches to Inhibit Botrytis cinerea

Botrytis cinerea is a pathogenic fungus that has tremendous adverse impact on agriculture around the world. With susceptibility from seed to storage, highly adaptive nature under selective pressure, and ability to thrive at lower temperatures, B. cinerea is a formidable challenge. Technological advances in genetics, biochemistry, and biotechnology are providing new and unparalleled possibilities fo r treatment and management. With the resulting outburst of novel antifungal agents, it is difficult to keep abreast. This review not only catalogs and summarizes recent antifungal discoveries that target B. cinerea, but tries to give the reader a feel for how expansive the new possibilit ies are. Due to practical limitation, this review focuses on antifungal peptides/proteins and antifungal natural products. A brief perspective on emerging antifungals that hold potential to significantly impact the botryticide landscape is also included.


Introduction
Botrytis cinerea is a high risk necrotrophic pathogen known to damage over 230 p lant species world wide [1].This fungus can infect agriculturally important food crops including fruits, vegetables, cereals and even ornamental plants costing billions in damages yearly.The severity of outbreaks is dependent upon the prevailing environ mental conditions.In severe epidemics, 100% yield loss can occur.B. cinerea can attack leaves, stems and fruit, often with heavy losses after harvest when crops are particularly vulnerable. Worse yet, it appears that B. cinerea enters a young host, remains quiescent, and then becomes active when the environment is conducive or plant physiology changes [1][2][3], Figure 1. B. cinerea is common in most climates manifesting itself as gray mold due to its property of producing a large number of spores.It sporulates profusely and dry conidia are dispersed through the air making this pathogen a constant threat to susceptible crops. Moreover, the ability of B. cinerea to be active at low temperatures (i.e. 0 ℃) [4] makes it a challenge for disease management during storage and shipment.
Chemical contro l by several families of fungicides has been standard practice for many years.Applied doses vary fro m 400 to 3000 g/ha and the number of treatments during a season ranges fro m one o r t wo , to mo re t han t wenty.
Treatmen ts o f s eed s o r b u lbs , as well as fu ng icid e applicat ions after h arvest are co mmon .The ab ility of B. cinerea to quickly adapt to new chemistries leads to the Life cycle of B. cinerea during strawberry infection. Conidiophores (A) are sources of dry conidia (B) that travel through air and can infect blossoms and leaves of a young plant (C).Direct infection of ripening or ripe fruit (D) is also possible.After season, B. cinerea overwinters in the rotten debris (E) to start growing again when conditions are favorable. Hyphae (F) that give rise to conidiophores As evidenced from existing antifungals, understanding of B. cinerea is paramount for durable and sustainable disease control.Therefore, before discussing new antifungals, it is important to understand the infection process.Most simply, fungal infection is co mprised of three main steps: 1) attachment and penetrating the host tissue/surface which involves lipases, cutinases, and pectinases; 2) killing of host cells including secreted toxins, o xalic acid, andinduction of active oxygen species; 3) conversion of host tissue into fungal bio mass.All of these steps can potentially be exploited to inhib it B. cinerea.Undoubtedly a combinatorial approach targeting mult iple steps with mu ltip le antifungals will be most effective.That means new targets and new antifungals will play significant roles.
Described in this review are t wo general classes of antifungals shown to be effective against B. cinerea infection, antifungal peptides/proteins and natural products (broadly defined).Earlier reviews have covered similar topics, so focus will be predo minantly on more recent reports.It should also be noted that other, in many cases related, reports of antifungal activity have been published. However, only those antifungals that have explicit ly shown activity against B. cinerea are included in this review.A lso it should be noted that direct comparison of inhibitory potency is difficult.In addition to multiple assay types, different parameters (IC 50 , MIC, qualitative results) are reported.Further compounding the problem, so metimes only mixtu res of natural products are available.W ith these limitat ions in mind, the authors refrain fro m terms such as highly active, very potent, strong inhibition, and the like when describing antifungal effects.Summary tables are included at the end of both major sections.

Antifungal Peptides and Proteins
Antifungal peptides and proteins are produced throughout the phylogenetic tree, fro m p rokaryotes through higher eukaryotes.These peptides and proteins have a variety of amino acid sequences and multip le databases provide informat ion about them [13][14][15].Historically, peptides and proteins have not been considered viable treatment candidates due to their costly manufacture.Advances in large scale recombinant expression (antifungal peptides and proteins made in bacterial or yeast systems and applied to crops in the field) and the growing transgenic possibilities (antifungal peptides and proteins expressed in modified crops) make peptide and protein biologics ever mo re feasible.As such, new research in the area has opened many new possibilities.Several rev iews are available to acquaint the reader with certain subsets of antimicrobial peptides and proteins [16][17][18][19][20].Th is review focuses on antifungal peptides and proteins that have demonstrated activity against B. cinerea.

Single Celled Organisms: Yeast, Fungi, and Bacteria
Single celled organisms are abundant producers of antifungal agents.Part of their arsenal is a range of antifungal peptides and proteins.Fro m the many antifungal peptides and proteins recently reported, those found active against B. cinerea are presented below.
Derived fro m a single-chain antibody and acting as a yeast killer to xin, the 10 amino acid killer pepti de was shown to exert act ivity against a broad spectrum of phytopathogenic fungi, including B. cinerea [21].Successful transgenic expression helped to demonstrate the killer peptide is a promising antifungal peptide.Expression in planta as a viral coat fusion protein using a potato virus system yielded chimeric virus particles displaying the peptide which exhibited enhanced antifungal act ivity.The authors further suggested that the potato virus system is an efficient method to produce and evaluate antimicrobial peptides in plants, an assertion that held true [22]and extended to human proteins.In fact, it appears that transient exp ression of foreign genes using plant viral vectors can produce immunogens impacting new vaccine development [23].
For some time, the saprophytic fungus Ulocladium atrum Preuss has shown promise as a b iological control agent for B. cinerea [24][25][26].More recently, the individual active antifungal components have been uncovered including a cyclope ptoli de [27].Th is unnamed cyclopeptolide was most potent against B. cinerea with more moderate act ivity towards other fungi.It should be noted that this cyclopeptolide was originally identified as an antifungal substance against yeasts and yeast-like fungi [28].However, this report was the first to demonstrate its activity against filamentous fungi.
Aureobasidin A is a cyclic depsipeptide produced by the yeast-like fungus Aureobasidium pullulansR106 [29].It reduces conidial germination rates, delays conidial germination init iation, restricts germ tube and myceliu m elongation, and induces abnormal mo rphology in germ tubes and hyphae.It has antifungal activity towards B. cinerea among other pathogenic fungi [30]and yeast [31].Though sensitivity was species dependent, 50 µg/ ml was effective in reducing the incidence and severity of gray mold on strawberries after art ificial inoculation [29].Aureobasidin A targets inositol phosphorylceramide synthase [32], an essential fungal en zy me absent fro m hu mans and other mammals.The gene encoding its biosynthesis complex has been characterized [33] and the high resolution structure solved [34]. A ided by an extensive background and supporting information, Aureobasidin A holds considerable promise as relatively safe crop antifungal.
Valinomycin is also a depsipeptide antagonistic towards B. cinerea.It has been purified fro m culture extracts of Streptomyces sp. (M10), a g ram-positive bacterial strain isolated fro m soil [35].It is a natural ionophore facilitating potassium ion movement through lipid membranes. Valino mycin showed in vitro antifungal activity against B. cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants.Disease control efficacy was similar to the co mmercial fungicide Vinclo zolin. Long known to have antifungal activ ity [35], this was the first report on disease control against Botrytis blight.The genes responsible for b iosynthesis are known [36] so transgenic implementation is possible.However, in planta expression may be limited by the supply of D-amino acids, though both enantiomers are found in plants [37,38].
Colletotrichum dematium is an endophytic fungus from Costa Rica that makes Colutellin A [39].Colutellin A has a mass of 1.1 kDa and contains isoleucine, valine, serine, N-methyl-valine and beta-aminoisobutryic acid in a mo lar ratio of 3:2:1:1:1, respectively.It was isolated fro m culture broth by tracking antifungal act ivity.At 48 hours post exposure, naturally expressed Collutellin A has a MIC of 3.6 µg/ml against B. cinerea.
The mo ld Aspergillus giganteus produces a basic low-mo lecular-weight peptide, generically designated AFP, that is structurally related to plant defensins and thionins [43].It has an appro ximate molecu lar weight of 5.5 kDa and is composed of 51 residues, 4 of wh ich are cysteine. It is secreted as an inactive precursor containing an amino-terminal extension of 6 amino acids which is later removed to produce the active protein.AFP consists of 5 antiparallel beta strands forming a compact beta barrel that is stabilized by disulfide bonds [44] and has recently been recomb inantly expressed in yeast [45].AFP inhibited B. cinerea with an IC 50 below 1 µM at 24 hours [43].Mycelial growth and conidial germination were inhibited and application to plant leaves protected against Botrytis infection.An additive effect against the fungus was observed when AFP was combined with Cecropin A, a membrane disrupting peptide.These results indicate the potential of AFP and the afp gene to enhance protection against B. cinerea.
Similar to AFP, a novel antifungal protein/peptide Ac AFP is produced by the fungus Aspergillus clavatus [40].In the genome, the 282 base pair open reading is interrupted by two small introns.The cDNA coding for AcAFP represents a pre-pro-protein of 94 residues that is processed to a 51 amino acid, cysteine-rich mature product.AcAFP has molecular mass of 5.8 kDa and is very thermostable.Structural modeling[42] suggests a compact fold of five anti-parallel beta sheets forming a barrel stabilized by four internal disulfide bonds.A putative cation binding site and adjacent hydrophobic stretch may be responsible for dose-dependent cell wall alterations.AcAFP inhibits mycelial gro wth of B. cinerea, amongst other pathogenic fungi.Reco mb inant AcAFP production has been optimized and conditions affecting antifungal activ ity determined [41].Activity decreased at high ionic strength and in the presence of 10 mM of divalent cations (Mn 2+ , Fe 2+ and Ca 2+ ).
Bacisubin is a 41.9 kDa antifungal protein isolated fro m cultures of gram-positive bacteria Bacillus subtilis strain B-916 [46]. Bacisubin exh ibited inhibitory act ivity on mycelial growth of B. cinerea (and mu ltip le other fungi) with an IC 50 value of 2.74 µM.Bacisubin function is unknown, though did demonstrate ribonuclease and hemagglutinating activities.
An alkaline protease ALP5 fro m the yeast-like fungus Aureobasidium pullulans PL5 was shown to play an important role in biocontrol of B. cinerea [47].A LP5 is composed of 415 amino acids, has a calculated molecular weight of 42.9 kDa and an isoelectric point of 4.5. Reco mbinant expression yielded active, ho mogeneous ALP5 that inhib ited mycelial growth of B. cinerea.
Another pair of proteins with antifungal potential are the BcSnod1 and BcSnod2 virulence factors fro m B. cinerea [48,49].These highly similar, ~13 kDa proteins are secreted by B. cinerea and have been shown to induce necrosis in plant hosts.The BcSnods are members of the Ceratoplatanin family o f s mall phytotoxic proteins, acting quite the opposite of defensins.However, studies have shown that low level transgenic expression of BcSnod related proteins confer increased resistance to B. cinerea in plant hosts [50]. Therefore a new "desensitizing" approach may be possible for these antifungal proteins.

Plants
Through their life cycle, plants employ mu ltip le means to counter fungal infection.Diverse plants fro m around the globe have been found to produce antifungal peptides and proteins.A subset of this vast array of antifungal peptides and proteins is active against Gray Mold and described below.
First isolated fro m potato over a decade ago [51], Snakin-1 is composed of 63 amino acids.It contains 12 cysteine residues, has a molecu lar weight of 6.9 kDa, and has some sequence motifs in common with kistrin and other hemoto xic snake venoms.Protein recomb inantly exp ressed in bacteria exh ibited antifungal act ivity against B. cinerea with an IC 50 of 5-14 µM, similar to original reports of activity fro m native plant derived protein [51,52].Snakin-1 was shown to be self-interacting, localizing in the plas ma memb rane and detailed analysis revealed effects on cell division, metabolis m, and cell wall co mposition. Though overexpressing Snakin -1 in potato did not cause morphological differences, transgenic studies have not been promising [53].
Ascalin was isolated from the bulbs of the shallot Allium ascalonicum [54].It has a molecular weight of 9.5 kDa with sequence similarity to chitinases from other Allium species. Ascalin demonstrated somewhat specific inhibit ion of mycelial gro wth in B. cinerea. with an IC 50 of 2.5 µM. Vulgin is another antifungal protein purified fro m pinto beans [55].Like Ascalin, Vulgin bears some sequence homology to chitinases.At 28 kDa, Vulgin is considerably larger than Ascalin but similar in size to jack bean and peanut chitinases. Unlike Ascalin, Vulgin exerted antifungal activity toward mult iple fungi including Mycosphaerella arachidicola, Fusarium oxysporum, and Coprinus comatus. Vulgin had the weakest inhibit ion against B. cinerea, with an IC 50 of 7 µM.
Li menin was isolated fro m shelf bean seeds [56].It has a mo lecular weight of 6.5 kDa and suppressed mycelial growth in B. cinerea, among other fungi, with an IC 50 of 2.9 µM.Its mechanism of action is not known, though inhibited translation in a eukaryotic cell-free system.Also fro m a legume, Mung oin is a novel protease inhibitor isolated from mung bean (Phaseolus mungo) seeds [57].It has a molecular weight of 10 kDa and displayed inhib itory activ ity toward a variety of fungal species including B. cinerea.No follo w-up publications were available for either of these antifungal proteins.From another legume, a 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activ ities, was isolated fro m the large lima bean (P. limensis) legumes [58]. Limy in's N-terminal amino acid sequence is highly homo logous to plant defensin and defensin precursors.Limy in is thermostable up to 80 ℃ and suppresses mycelial growth.No IC 50 was reported for B. cinerea.These three examp les perpetuate the axio m that legumes produce an abundance of proteins and peptides with important biological act ivities, including a nu mber of antifungal peptides and proteins [16].
Ganoder mi n, a 15 kDa antifungal protein, was isolated fro m the medical mushroom Ganoderma lucidum [59].The N-terminal sequence of Ganodermin somewhat resembles Lyophyllum antifungal p rotein and Eryngin with slight similarity to thaumatin and thaumatin -like proteins noted.It inhibited mycelial gro wth of B. cinerea, among several fungi, with an IC 50 value of 15.2 µM.The mechanism of antifungal inhibit ion is not known, though common activities including hemagglutinating (lect in), deo xyribonuclease, ribonuclease or protease inhibition were not observed.
An antifungal protein with a mo lecular mass of 26.9 kDa was isolated fro m dry seeds of the foxtail millet Setaria italica (L.) Beauv. [60].The unnamed protein inhibited mycelial growth and exhib ited antifungal activity against B. cinerea among other fungi.Observations from light and atomic force microscopy indicated that the mode of antifungal activity centered on attack on the fungal cell wall and retraction of cytoplasm in the hyphae leading to death of the myceliu m.The modificat ion caused by the foxtail millet antifungal protein may be related to interference of cell wall synthesis, affecting fungal morphogenesis and growth.
Osmotin and another Thaumatin-like protein were isolated fro m grapevine (Vitis vinifera) [61].Both exh ibited antifungal activity against B. cinerea.Upon fungal infection in grapevine leaves, both are induced and accumu late in the leaves and berries.These relatively well established antifungal proteins blocked mycelial g rowth, inhibited spore germination and germ tube growth of B. cinerea.Thaumatin and Thaumatin-like proteins have been transgenically expressed, furthering their progress as biocontrol agents.A notable finding was that in combination, these antifungal proteins display synergism [61] possibly providing insight into the rather wide range of fungal processes thaumatin-like proteins are involved in [62].Osmotin is a ho molog of mammalian adiponectin that induces apoptosis in yeast [63]. It exerts its biological activ ity through mamma lian adiponectin receptor homologs that are part of a RAS2/ cAMP signaling pathway [63,64], in part weakening defensive cell wall barriers [65].Th is adds another dimension to the use of Osmotin in that it can be combined with other cell wall damag ing antifungal agents for potentially increased efficacy.
Pomegranin, isolated from fresh pomegranate peels, is an 11 kDa protein that inhibits mycelial gro wth in B. cinerea with an IC 50 of 2 µM [66].The N-terminal amino acid sequence resembles a rice disease resistance nucleotide binding leucine-rich repeat (NB-LRR)-like protein.Similarly, two novel antifungal proteins that showed sequence homology to NB-LRR proteins were isolated and characterized fro m rosemary pepper (Lippia sidoides Cha m.) flowers [67].They were 10 and 15 kDa in size and were ab le to inhibit B. cinerea develop ment.Many highly variab le NB-LRR disease resistance proteins are encoded in plant genomes.Also, plant NB-LRR immune receptors recognize a range of effector proteins fro m different pathogens and are incredibly adaptive in pathogen recognition and defense initiat ion [68].A growing body of evidence suggests the N-termin i of NB-LRR proteins also function in pathogen recognition [69].Directly applicable, Po megranin may be important in defining pathogen recognition specificity.It also appears that two NB-LRRs can function together to mediate disease resistance and only frag ments of NB-LRR proteins are sufficient to initiate defense signaling [70].It should be noted, however, that the NB-LRR frag ment sufficient for function is distinct between different NB-LRRs. Following the mult iple redundant theme, four defensins designated Hc AFP1-4 have been isolated fro m the flowering plant Heliophila coronopifolia, a native South African Brassicaceae species [71].Overall the peptides were 72% similar.However HcAFP-1 and -3 shared 94% ho mology and were unique in the Brassicaceae defensins.HcAFP2 and 4 were similar with Arabidopsis and Raphanus defensins. Ho mology modeling showed that the variable amino acids between the four HcAFP peptides altered surface properties. Moreover, the variability is located in regions thought to be responsible for determining specific act ivit ies for defensins. Activity against B. cinerea included membrane permeab ilization, hyper-branching, and bio mass reduction. Related to the similarity grouping, HcAFP2 and 4 caused memb rane permeabilizat ion whereas HcAFP1 and 3 caused mild morphogenetic effects, without any indication of memb rane activity.Moreover, exp ression is tissue-specific and similarly grouped with HcAFP1 and 3 exp ressed in leaves, stems and flowers, whereas HcAFP2 and 4 exp ressed in seedpods and seeds.This suggests protective roles in unknown developmental and physiological processes. Reco mbinant HcAFP2 and 4 peptides showed IC 50 values of 5-20 µg/ ml, whereas HcAFP1 and 3 were less potent.
Three novel ethylene response factor genes, BkERF1, BkERF2.1 and Bk ERF2 .2, were isolated fro m the med icinal plant Bupleurum kaoi [72].They are ubiquitously expressed at low levels in all parts of mature p lants, with BkERF2.2 moderately expressed in vegetative tissues. BkERFs contain a nuclear localization signal, an ERF/AP2 DNA binding do main and function as transcriptional activators.Transgenic overexpression of BkERFs enhanced resistance to B. cinerea providing evidence that BkERFs med iate the expression of defense-related genes in plants. ERF proteins are thought to integrate signals fro m jas monic acid, ethylene, and salicy lic acid defense regulated pathways [73,74].
The salicylic acid regulatory gene HopW1-1-interacting3, or WIN3, controls broad-spectrum d isease resistance to B. cinerea [75]. Prev iously shown to confer resistance to the Pseudomonas syringae, WIN3 acts additively with several salicylic acid regulators in regulating salicylic acid accumulat ion, cell death, and/or disease resistance.In Arabidopsis, salicylic acid med iated signaling is required for local resistance to B. cinerea [76]. Surprisingly, WIN3 affects flowering time and thus represents a novel node in salicylic acid signaling network regulat ing defense and development. It makes sense that immunity and development are connected, regulation of both being important for plant fitness.

Other Higher Organisms
Some h igher order organisms also produce a wide array of other defense peptides and proteins.Those tested and showing activity against B. cinerea are described below.
Human beta-de fensin-2 is a small peptide with broad antimicrobial activ ity.Transgenic exp ression in A. thaliana was shown to impart increased resistance to B. cinereain vitro and the resistance was correlated to the level of hu man beta-defensin-2 p roduced [77].Fro m known shared structural homology between human beta-defensins and plant defensins, functional ho mology was demonstrated between plant and mammalian defensins kingdoms for the first time.Th is opens the possibility of transgenic antifungals in plants to antifungals from other eukaryotic kingdoms.Such a cross species approach could be quite effective for furthering antifungal strategies.
Ap is a 47 residue peptide of molecu lar weight 5.1 kDa isolated fro m the Chilean sea scallop Argopecten purpuratus hemocytes [78]. Based on the native sequence, a 30-residue synthetic peptide, Ap-S, was designed, that was much more active than native Ap.Ap-S has been expressed recomb inantly and no cytotoxicity was observed in fish [79]. The recombinant protein inhibited B. cinerea growth at 81 nM by affecting hyphae structures and spore count.
Three wasp venom peptides isolated fro m Orancistrocerus drewseni (Hy menoptera: Eu men idae) exhibited antifungal activit ies [80]. These peptides, designated OdVP1-3, contain a high content of positively charged and hydrophobic amino acids indicative of amphipathic alpha-helices. All three share typical characteristics of amidated C-termini proteins. Of note, amidizat ion neutralizing negative charge on the C-terminus is a common posttranslational modification in peptide hormones and also found in mu ltip le redundant neurotoxins isolated fro m snake veno m [81].Od VP2 and a variant with two additional N-terminal amino acid residues showed the most potent antifungal activity, with IC 50 values near 0.5 µg/ml. Table   Table 1

Antifungal Natural Products
Plants possess a wide array of defense mechanisms and defensive countermeasures against pathogenic fungi.One category already discussed is the antifungal peptides and proteins.Another is the small mo lecule natural products. Generally termed secondary plant metabolites, this broadly defined category can be divided into numerous subsets based on source, chemistry, inhibit ion mechanism, etc.In this review, they are divided into biosurfactants, essential oils, volatile co mpounds, and other natural products.As with the antifungal peptides and proteins, many new small molecu le natural products have been found to have antifungal activity in recent years.Some are modified versions of past successes and others represent new classes of mo lecules altogether. Those with reported activ ity against B. cinerea are included below.

Biosurfac tants, Li pope pti des, and Li pi d Relate d Anti fung als
Biosurfactants are amph ipathic detergent-like mo lecules produced from natural sources.Popularity and interest in biosurfactants come fro m their widespread applicability, diversity, selectivity, low to xicity, environ mentally friendly nature (biodegradability, bioco mpatibility), availab ility fro m large-scale production, and relative ease of preparation. Included in this section are the lipopeptides which are related to both biosurfactants and the antifungal peptides. Most are of natural origin though synthetic biosurfactants are also included.
The rhamnoli pi ds are simple g lycolip ids comprised of a fatty acid tail with one or two rhamnose rings at the carboxyl end of the fatty acid (see Figure 2A).Their antifungal activity comes fro m disruption of fungal cell memb ranes.Zoospores are especially vulnerable to rhamnolipids because they lack the protective cell wall present in other fungal life stages.Rhamnolipids produced by the gram-negative bacteria Pseudomonas aeruginosa triggered defense responses and protection against the fungus B. cinerea in grapevine [83] by inhibited spore germination and myceliu m growth. Another Pseudomonas strain, Pseudomonas putida strain 267, also produces biosurfactants that lyse zoospores [84]. These biosurfactants, identified as cyclic lipope pti des resemb ling putisolvin, were also shown to inhibit B. cinerea growth.
Isolated from fermented food, a gram-positive Bacillus subtilis strain was found to produce biosurfactants with antifungal activity [85].The biosurfactant activity was stable at high temperature and a wide range of pH and salt concentrations.In a subsequent report, this Bacillus subtilis strain was shown to inhibit myceliu m gro wth of B. cinerea by 70%.Further analysis of active co mponents lead to the characterizat ion of a mixture lipope pti des and li pope pti de homologs [86].
Another mixtu re of antifungal lipopeptides was isolated fro m a gram-negative Acinetobacter baumanniistrain [87].It was found that three antifungal lipopeptides were secreted, all isolated and identified as isomers of Iturin A (a pore-forming lipopeptide).Indiv idual IC 50 values were not reported though 50% gro wth inhib ition of B. cinerea was observed at 25 µl of filtrate per milliliter of fungal cu lture.
The cyclic lipopeptides Neope ptin A and B were isolatedfrom the culture broth of fro m g ram-positive actinobacteria Streptomyces sp. KNF2047 [88](See Figure  2B). Neopeptin A and B demonstrated inhibitory activities against mycelial growth of B. cinerea and other pathogenic fungi. At a concentration of 2.4 mg/ L, their disease control activity was 92.1% and a mixture of these Neopeptins showed protective and curative activity against cucumber powdery mildew in vivo.It should also be noted, that these were in itially characterized and the structure solved over 25 years ago [89].
New antifungal agents have also been developed from microbial modification of pol yunsaturated fatty aci ds. Cultures of Pseudomonas aeruginosa PR3 have been used to make docosahexaenoic acid, which inhibited mycelial growth and had a strong detrimental effect on spore germination in B. cinerea [90]. Min imu m inhibitory concentrations are in the range of 125-500 µg/ ml.  [91].B. cinerea infection was prevented by co-inoculation with wild-type U. maydis sporidia.Having identified the gene cluster that codes for ustilagic acid biosynthesis, it was shown that U. maydis mutants defective in ustilagic acid biosynthesis did not inhibit B. cinerea infection. Though ustilagic acid was not isolated and tested individually for antifungal activity, it appears that this biosurfactant plays an integral role in U. maydis interruption of B. cinerea infect ion.
Alkami des are fatty acid amides of wide d istribution in plants. N-isobutyl decanamide application significantly reduced necrosis caused by B. cinerea and inhibited fungal proliferation [92].It appears that alkamides modulate certain necrotrophic-associated defense responses.
Synthetic li popepti des composed of four amino acids lin ked to fatty acids were shown to induce systemic fungal resistance in plants [93].In particular, two synthetic lipopeptides (C16-KKKK and C16-KLLK wherein the third residue is a d-enantiomer) showed inhibition.These lipopeptides induced expression of defense-related genes in planta. Thus, short cationic lipopeptides are emerging as new putative antifungal agents.

Essential Oils and Volatile Compounds
In addition to antifungal peptides/proteins and biosurfactants, essential oils and volatile co mpounds are another category of small mo lecule natural products with antifungal activity.Bioactivity in the vapor phase makes volatiles very attractive as possible postharvest fumigants. Interest is especially gro wing in the use of naturally derived essential oils [94] and volatile co mpounds as fungal control agents in agriculture, fueled by safety concerns regarding synthetic antifungals.To distinguish from vo latile compounds, essential oils are the odorous and volatile products of plant secondary metabolism.Essential oils appear to have evolved to protect plants against invading pathogens [95].Widely used in traditional/folk medicine, their antifungal activ ity is well documented [96].Becoming somewhat dated, a past review covers antibacterial and antifungal activities of essential oils and their mechanisms of action [97]. As before, only more recently reported volatiles and only those that demonstrate activity against B. cinerea will be covered.Grouped in the categories of essential oils and volatile co mpounds, further subdivision is based on source.

Essential Oils
Fro m the fragrant nature of many herbs and spices, it is not surprising that they possess a variety of essential oils.It has long been known that certain spices and herbs have strong antiseptic properties.Moreover, ext racts or co mpounds found in the ext racts of numerous spices and herbs have been reported to show activity against B. cinerea.They are described below according to the plant origin.
Lamiaceae, Verbenaceae: Fro m Moroccan herbs of the Lamiaceae family, essential oils fro m Origanum compactum and Thymus glandulosus completely inhib ited B. cinerea at 100 pp m with IC 50 values of 35.1 and 79.2 pp m, respectively [98].The two main constituents, thymol and carvacrol, exh ib ited the strongest antifungal activity with complete inhibition for both at 100 pp m.These volatiles inhibited the growth of the myceliu m.Essential oil of Mentha pulegium showed moderate inhib ition with IC 50 of 233.5 pp m.Essential oils obtained fro m other Lamiaceae fa mily me mbers (Origanum syriacum L. var. bevanii, Lavandula stoechas L. var. stoechas, and Rosmarinus officinalis L.) were also found to have activity against B. cinerea [99]. Different concentrations inhibited growth in a dose-dependent manner and the volatile phase was consistently more effective than the contact phase.Vo latile vapor fro m Origanu m o il co mp letely inhibited B. cinerea growth at 0.2 μg/ ml air whereas the essential oils of lavender and rosemary required concentrations of 1.6 μg/ ml air.For the contact phase, the concentration of Origanu m oil had to be 12.8 μg/ ml to inhibit the growth of B. cinerea completely.Rosemary and lavender essential oils required roughly twice the concentration for complete inhibit ion in the contact phase.These essential oils cause morphological degeneration of hyphae including shriveling, cytoplasmic coagulation, vacuolations, and loss of conidiation.Spore germination and germ tube elongation were also inhib ited.In vivo assays resulted in protection of tomato against B. cinerea.
Essential oils fro m Mentha piperita (peppermint) and Lavendula angustifolia (lavender) also demonstrated antibotrytis activity [100].The strongest antifungal activity was fro m L. angustifolia, completely inhibiting B. cinerea at 1,000 pp m with an EC 50 of 311.24 pp m.Similarly, essential oils fro m three Mediterranean aro matic plants (Verbena officinalis, Thymus vulgaris and Origanum vulgare) were found to harbor activity against phytopathogenic fungi [101]. In fo llo w-up studies, the eight main co mponents of these essential oils were tested against agents of post-harvest fruit decay including B. cinerea [102].Citral and carvacrol at 250 ppm and thymol at 150 pp m stopped B. cinerea growth.
Asteraceae: Essential oils fro m several flowering plants have demonstrated antifungal activity against B. cinerea. Essential oil fro m the Indian marigold Tagetes patula, in the daisy family of Asteraceae, completely inhib ited B. cinerea growth at 10 µl/ ml [103].A mu ltisite mechanism of action is proposed since ultrastructural modifications in mycelia and large alterations in hyphal morphology were observed.Also, the effects could not be attributed to any one of the two major components of the essential oil (piperitone and piperitenone). The effects could be a result of synergism between different chemical characteristics of the essential oil.In a related report, essential oils and two pure compounds (carvacrol and beta-bisabolol) fro m three different flowering Asteraceae demonstrated antifungal activity [104]. Subsequent evaluation indicated that alpha-bisabolol was weakly responsible for part o f the B. cinerea growth inhibit ion.At a higher concentration, 1,000 mg/ mL, essential oil of Artemisia argyi Lévl. et Vant inflorescence (wormwood) was also shown to inhibit B. cinerea growth [105].
Apiaceae, Myrtaceae: Pimpinella is a genus in the Apiaceae family, co mmonly referred to as the carrot or parsley family.A new 'phenylpropanoid', 4-(3-methyl-o xiran-2-yl) phenyl 2-methylbutanoate was isolated from the essential oils of Turkish Pimpinella species [106].This co mpound together with epoxypseudoisoeugenyl 2-methyl-butyrate demonstrated growth inhibit ion against B. cinerea with IC 50 values of approximately 0.3 µM and 30 µM at 48 hours for the phenylpropanoid and epoxy-butyrate, respectively.
Fro m a screen of fifty-t wo herbs and spices, volatiles fro m black zira exhib ited the strongest inhibition of B. cinerea, followed by cu min and cardamo m [107].Seven volatile compounds from b lack zira were detected with cuminaldehyde and p-cymene begin the most potent botryticides. Though EC 50 s were 99.7 pp m for p-cy mene and 0.0312 pp m for cu minaldehyde against F. oxysporum, no values were reported for B. cinerea.Essential oils the Myrtaceae Syzygium aromaticum (cloves) and the Apiaceae Foeniculum vulgare (fennel) showed nearly identical antifungal activity against B. cinerea [108].The effects were dose dependent with 85% inhibit ion at 1 µL/ mL after 10 days dropping to approximately 50% after 20 days for both.
Caryophyllaceae: Antifungal activity was also reported for essential oil and organic extracts isolated fro m the floral parts of Silene armeria L. of the carnation family [109].B. cinerea growth inhibit ion ranged of 39.6-67.6%, along with their respective MIC values ranging fro m 62.5 to 1000 µg/ ml. Strong detrimental effects on spore germination were observed.

Volat ile Co mpounds
Antifungal volatile co mpounds are very much related to essential oils, usually being derived fro m them.The major difference is that volatile co mpounds are individual compounds whereas essential oils are often uncharacterized mixtu res.
Vol atiles from Is abella grapes were shown to reduce the inoculum and pathogenicity of B. cinerea [110,111]. Inhibitory action was on sporulation and sclerotia formation of the fungus, with pronounced inhibition at cooler temperatures. The active co mponents were not identified and no follo w-up has been published.Another antifungal volatile compound commonly found in essential oils is allo-oci me ne. This compound was shown to enhance resistance of Arabidopsis thaliana against B. cinerea [112,113].Hyphal penetration and growth were suppressed and allo-ocimene induced lignification and accu mulation of antifungal substances, in particular camalexin .Moreover, allo-ocimene treatment resulted in more rapid and more intense responses to B. cinerea challenge, suggesting allo-ocimene primes defensive responses [113].
The C6-al dehydes are another family of volat ile compounds with antibotrytis activity [113].They are made by hydroperoxide lyase (HPL) and co mmon to most terrestrial p lants.Arabidopsis thaliana with upregulated HPL produced large quantities of these volatiles that were found to inhibit B. cinerea infection.This effect was proportional to the level of expression of HPL suggesting a direct fungicidal activ ity of C6-aldehydes.
With the potential utility of volatile co mpounds as antifungals, the search for volatile co mpounds has expanded beyond plants. Other sources including bacteria and fungi have been reported to produce antifungal volatile compounds. 5-pentyl -2-fural dehyde was isolated from cultures of the fungus Oxyporus latemarginatus EF069 [114]. Purified 5-pentyl-2-furaldehyde inhibited mycelial growth of B. cinerea in a dose-dependent manner. Mycofumigation with solid EF069 cu ltures reduced the development of postharvest B. cinerea in apples demonstrating the possible use of either Oxyporus latemarginatus EF069 or 5-pentyl-2-fu raldehyde as a biocontrol agent.
Another group of fungi, the Chilean saprobi ontic fungi, were shown to have antifungal activity [115].Of those fungi tested, Schizophyllum commune emitted volatiles that exhibited the highest inhibitory activity against B. cinerea.
Vo latiles produced by the yeast Candida intermedia strain C410 suppressed B. cinerea growth [116].The two most abundant compounds, 1,3,5,7-cyclooctatetraene and 3-me thyl-1-butanol, were h ighly inhibitory to conidial germination and mycelial growth. It was also shown that the incidence and severity of Botrytis fruit rot on strawberry was reduced by exposure of strawberry fru it to these volatiles, demonstrating biofumigant control fo r a vo latile compound.

Other Natural Products
In addition to essential o ils and vo latile co mpounds, several other small mo lecule natural products are deserving of mention.Tea pol yphenols were reported to control Gray Mold in grape [118]. They significantly inhibited B. cinerea spore germination at h igher concentrations and inhibited myceliu m growth at lower concentrations. Glyceollins are a novel class of antiestrogenic phytoalexins fro m soybean. They had detrimental effects on B. cinerea spore germination, though no quantitation was reported [119]. Sixteen compounds isolated from the fermentation broth of the endophytic fungus Aspergillus fumigatus LN-4 showed antifungal activit ies [120]. Four of the co mpounds (12β-hydroxy-13α-methoxyver-rucul ogenTR-2, fumitre  -morgin B, verrucul ogen, and hel volic aci d), exh ibited antifungal activit ies with MIC values of 6.25-50 μg/mL.The structures of all co mpounds were determined and structureactivity relat ionships discussed in this report.

Emerging B. cinerea Antifungals
Advances in biotechnology coupled with pathogen specific knowledge propel new antifungal approaches.What follows is a rapid fire summary of emerging possibilities that fall outside the previously discussed categories.The first section focuses on B. cinerea intracellular signaling, with better understanding of cellular pathways leading to increased potential for antifungal d iscovery.The second part centers on transgenic possibilities.The remainder deals with other prospects worthy of mention, but again not fitting in previous sections.

Insight into B . cinerea Intracellular Signaling with Potential for Intervention
A good examp le for fungicide development based on understanding the intracellular signaling is the phenylpyrrole fungicide Fludi oxonil.It activates the osmotic MAP kinase cascade via Hog1 (High osmolarity g lycerol response) [121]. In Neurospora crassa, the Hog1 pathway regulates expression of a transcription factor downstream of the OS-2 MAP kinase involved in conidia stress tolerance [122].It is suggested to regulate genes involved in conidia format ion, circadian rhythm, and ascospore maturat ion.A large number of genes dependent on the Hog1 MAPK cascade are modulated by Fludio xonil [123].In response to osmotic stress and Fludio xonil, expression of genes for glycerol synthesis (gcy-1, gcy-3, and dak-1), gluconeogenesis (fbp-1 and pck -1), and catalase (ctt-1) are activated [124].Fludio xon il also causes a plasma memb rane hyperpolarizat ion and H + e fflu x, consistent with activation of the H + -ATPase. Concomitantly K + uptake occurs and turgor (pressure that pushes the plasma memb rane against the plant cell wall) increases about 2-fo ld above normal levels [125].
Better understanding of intracellular signaling has led to additive measures for antifungal t reat ment.Again in N. crassa, histidine kinase mutations conferred Fludio xon il resistance and osmotic sensitivity [126].Therefore a histidine kinase activating compound could be used to increase the longevity of Fludio xonil potency and delay onset of resistance development.In Cryptococcus neoformans, Hrk1 is analogous to Hog1.A hrk1Δ mutant exhibited almost complete resistance to Fludioxonil [127]. Interestingly, inhibit ion of Hrk1 substantially increasing azo le drug susceptibility, provid ing another novel strategy for combin ing antifungals.
Coapplication of 2,5-dihydro xybenzoic acid, an aspirin/salicylic acid metabolite, elevates the activity of Fludio xon il through disruption of cellular g lutathione reduction/oxid izat ion homeostasis [128]. Fludio xon il activity could also be enhanced by using berberine and phenolic co mpounds to target antioxidative stress response in fungi [129] or natural chemosensitizing agents targeting the oxidative stress-response system [130]. Redo x-active compounds can serve as chemosensitizing agents to overcome resistance and lower effect ive fungicide dosages, reducing costs while lo wering of environ mental and health risks.
Another emerging antibotrytis prospect centers on stress response processes.The ability to adapt to mult iple stresses requires cross-talk and fine-tuning of stress signaling pathways. For pathogen signaling, several new interconnections have been uncovered. BcSak1 is a mitogen-activated protein kinase found in B. cinerea.The majority of genes regulated by BcSak1 are not involved in stress responses [131], however BcSak1 is involved in regulation of secondary metabolis m with secreted phytotoxins reduced in deletion mutants.In planta experiments underlined the essential role of BcSak1 in the early stages of infection, showing it translocates to the nucleus and then changes to cytosolic distribution during hyphal growth within the tissue.Historically kinases are good drug targets, pointing to favorable possibilities for BcSak1 intervention.
Another potential point for intervention is through Atf1-ho mologous basic region leucine zipper transcription factors. These act downstream of stress-activated mitogen-activated protein kinases and regulate transcription of general stress response genes.In B. cinerea, BcAtf1 is connected to the BcSak1, sharing stress response target genes [132]. Like BcSak1, BcAtf1 regulates a diversity of cellu lar processes including differentiation, though their roles are opposing.BcAtf1 knockout mutations also increased sensitivity to cell wall-interfering agents suggesting additional antifungal applications.
Also involved in intracellular signaling are Bc pp2 Ac (a catalytic subunit of a PP2A serine/threonine protein phosphatase) and SPT3 (a subunit of the Spt-Ada-Gcn5-Acetyl-transferase-like transcriptional regulator complex). Gene rep lacement and silencing approaches revealed that both are crucial for viru lence, growth, and differentiation as well as for resistance to peroxide in B. cinerea [133].Their critical functions point to these genes as possible targets for antifungal development.

Transgenic
Introducing another organism's defense mechanisms into plants to increase protection against B. cinerea is a growing means of engineering resistance.Genetic modification does face societal reluctance in accepting modified crops. However, part icularly pro mising is the transgenic expression of antifungal peptides or proteins since disruptions of normal metabolite flo w are unlikely. Regardless, progress with transgenics continues to advance many types of disease resistance in plants, including antifungal development.
Tobacco and tomato plants transformed with the Inhi bitor of Virus Replication (IVR) gene become partially resistant to B. cinereainfection [134,135]. IVR-like compounds were found in the certain species that were highly resistant to certain v iruses.Plant protein extracts containing transgenically expressed SG2 chitinase fro m Bacillus pumilus displayed a high inhibitory effect on spore germination and hyphal gro wth in B. cinerea [136]. Antifungal activity of truncated SG2, lacking the chitin binding and fibronectin type III domains, was lost.Both forms showed essentially equal hydrolytic activity toward colloidal chit in.These findings demonstrate that chitin binding and fibronectin type III domains play an important role in hydrolysis of chitin -glucan co mplex of fungal cell walls.Fro m Gerbera hybrida (Asteraceae), a newly cloned chalcone synthase-like polyketide synthase, 2-pyrone synthase was shown to be critical for inhib iting fungal infection [137].2-pyrone synthase is able to synthesize 4-hydro xy-6-methyl-2-pyrone (triacetolactone), a putative precursor for t wo abundant glucosides in gerbera.Gerbera plants lacking this gene are susceptible to B. cinerea infection and fail to produce several co mpounds induced upon infection with the mold.This synthase along with other members of the antifungal pathway could be used to generate fungal resistance with little to xicity expected from the new co mpounds introduced to the system for the Asteraceae plant family or others with similar pathways for phytoalexin generation.
Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2. Arginases catalyze breakdown of arginine into ornithine and urea. Arabidopsis plants overexpressing arginase were less susceptible to B. cinerea and accumulation of arginase-encoding gene mRNA was observed in Arabidopsis upon inoculation with B. cinerea [138].These results provide new insights into amino acid metabolic changes under stress and another potential mechanis m to disrupt B. cinerea.Believed to be involved in plant defense responses, lip id transfer proteins (LTPs) are members of the pathogenesis-related proteins (PR-14) family.A novel gene Ltp 3F1 encoding an antifungal protein fro m wheat (Su mai 3) was reco mb inantly expressed in bacteria [139].The LTP fusion protein exhibited a broadspectrum antifungal act ivity including activ ity against B. cinerea.Transgenic expression resulted in tobacco plants with normal phenotype and detached leaves showing increased fungal resistance.The demonstrated transgenic effects and potential for b road-spectrum antifungal act ivity make this LTP a v iable candidate for large scale use in crop plants.
Another tool to advance transgenic antifungals are wound-induci ble promoters, especially useful to limit production/accumulation of antimicrobial peptides and proteins to infected areas.A wound-inducible pro moter was reported for transgenic expression of a new ribosomeinactivating protein [140].Direct antifungal to xicity and reduced B. cinerea leaf damage was observed.

Miscellaneous
Not directly fitting into any previously described category, there are still several antifungal develop ments that are worth mentioning.They are listed chronologically here.
A new discovery has added to understanding of salicylic acid and its role in resistance.The enhanced pseudomonas suscepti bility gene, EPS1, was isolated and found to encode for a BAHD acy ltransferase [141].Mutations of EPS1, similar for other genes important for salicylic acid accumulat ion or signaling, impart enhanced resistance to B. cinerea.The authors suggest there is natural variation among the host (Arabidopsis ecotypes) with respect to the antagonistic cross-talk between defense signaling pathways against various types of microbial pathogens.Further characterizat ion of factors involved in the defense pathways will be needed to explain the mo lecular basis for the natural variation among different species.
Of larger perspective, a mu ltiobjective optimizat ion of the global antifungal profiles established a filtering strategy for new antifungal candidates.
Nanosized silica hybri d silver complex (NSS), nanosilver bound to silica molecules, also showed strong antifungal activity [143].The growth of Rhizoctonia solani was decreased by more than 90% at 6 µg/ ml of NSS added directly to the growth med ia.The antifungal effects were shown against B. cinerea, fro m wh ich the antifungal mechanis m o f the co mp lex was also determined.NSS solutions maintained stable antifungal activity for at least two years. Jasmonic aci d is a defensin that in recent years has become known fo r its ability to impart disease resistance.Plants grown fro m seeds treated with jas monic acid showed increased resistance against B. cinerea [144].Priming responses were long-lasting, with significant increases in resistance sustained in plants grown fro m treated seed for at least 8 weeks, and were associated with enhanced defense gene expression during pathogen attack. Long-term defense priming by seed treatments was not accompanied by reductions in growth, and may therefore be suitable for co mmercial applications.
Several new targets have also emerged for botryticide intervention.The Arabidopsis histidine kinase 5, known to med iate stomatal responses to exogenous and endogenous signals in Arabidopsis thaliana, contributes to B. cinerea resistance [145].Therefore upregulation in Arabidopsis or transgenic expression in another host may provide a means of increasing resistance to B. cinerea.Similarly, secreted papain-like cysteine proteases are important in plant immun ity. One such protease, RD21, was identified in tomato as crit ical for susceptibility to B. cinereainfection [146].Decreased expression or elimination of this gene product again could provide a means of limiting B. cinerea infectivity if altering RD21 does not disrupt normal cellu lar functions in tomato.

Conclusions
With fungicide-resistant strains, demand to reduce pesticide use, and appearance of iatrogenic diseases, there is clear need to develop new and alternative methods for managing B. cinerea as well as other pathogenic fungi.Fro m the growing wealth of informat ion, such approaches are being realized for every step of agricultural production. Priming of seeds, biocontrol and transgenic expression of any number of resistance enhancing genes during growth, and post-harvest examples have all been provided.To gauge the widespread development of antifungals, one only need look at the journals referenced in this rev iew.
Even with these advances, control of B. cinerea remains extremely difficult.In many cases, rapid adaptation to antifungal co mpounds occurs.Moreover, the broad habitat range, different infection strategies that vary along with conditions,ability to attack crops at almost any stage of growth, and ability to affect all parts of a plant contribute to the tremendous flexibility of this pathogenic fungus.The large toolbo x of en zy mes and metabolites that B. cinerea exploits to overco me host defenses adds to the difficulty.As witnessed for antifungal agents reported here, even rather ineffective agents are potentially vastly more potent and efficacious when used in comb ination.In fact, increased reliability and decreased variability in treat ment outcomes have already been shown for co mbinations of control agents [147]. On ly with continued advances including knowledge of the biology and epidemio logy of B. cinerea along with new antifungal development will the pro mise to safeguard agricultural productivity for generations to come be met.