Phytoalexin Formation Research Articles

Comparative study of the efficiency of inducers of cotton resistance to verticillium wilt

The effect of pre-sowing seed treatment with immunostimulant Bisol-2, red light and low frequency electromagnetic field on the content of fungitoxic substances of phenolic nature – phytoalexins (isohemigossypol and gossypol-equivalent) in infected etiolated cotton seedlings of S-4727 cultivar infected with Verticillium wilt pathogen was studied. It was found that photostimulation of seeds by red light induces phytoalexin formation in cotton tissues infected by the pathogen 1.5–2 times more effectively in comparison with Bisol-2 preparation or inducer of electromagnetic nature. The correlation between the content of phytoalexins in the tissues of seedlings, parameters of induction curves of chlorophyll fluorescence and the number of plants with signs of wilt lesions grown from treated and untreated seeds with inducers was revealed. This indicates the possibility of using red light and weak low-frequency electromagnetic fields as factors contributing to the intensification of phytoalexin formation in response to Verticillium wilt infection of cotton.

Multi-omics revealed molecular mechanism of biphenyl phytoalexin formation in response to yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells.

Yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells leads to the biosynthesis of various hormones, which activates specific signaling pathways that augments biphenyl phytoalexin production. Pathogen incursions pose a significant threat to crop yield and can have a pronounced effect on agricultural productivity and food security. Biphenyl phytoalexins are a specialized group of secondary metabolites that are mainly biosynthesized by Pyrinae plants as a defense mechanism against various pathogens. Despite previous research demonstrating that biphenyl phytoalexin production increased dramatically in Sorbus aucuparia suspension cells (SASCs) treated with yeast extract (YE), the underlying mechanisms remain poorly understood. To address this gap, we conducted an in-depth, multi-omics analysis of transcriptome, proteome, and metabolite (including biphenyl phytoalexins and phytohormones) dynamics in SASCs exposed to YE. Our results indicated that exposure to YE-induced oxidative stress in SASCs, leading to the biosynthesis of a range of hormones, including jasmonic acid (JA), jasmonic acid isoleucine (JA-ILE), gibberellin A4 (GA4), indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA). These hormones activated specific signaling pathways that promoted phenylpropanoid biosynthesis and augmented biphenyl phytoalexin production. Moreover, reactive oxygen species (ROS) generated during this process also acted as signaling molecules, amplifying the phenylpropanoid biosynthesis cascade through activation of the mitogen-activated protein kinase (MAPK) pathway. Key genes involved in these signaling pathways included SaBIS1, SaBIS2, SaBIS3, SaPAL, SaB4H, SaOMT, SaUGT1, SaLOX2, SaPR1, SaCHIB1, SaCHIB2 and SaCHIB3. Collectively, this study provided intensive insights into biphenyl phytoalexin accumulation in YE-treated SASCs, which would inform the development of more efficient disease-resistance strategies in economically significant cultivars.

Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Formation and exudation of biphenyl and dibenzofuran phytoalexins by roots of the apple rootstock M26 grown in apple replant disease soil

Apple replant disease (ARD) is a severe soil-borne disease frequently observed in apple tree nurseries and orchards worldwide. One of the responses of apple trees to ARD is the formation of biphenyl and dibenzofuran phytoalexins in their roots. However, there is no information on whether or not these phytoalexins are exuded into the soil. To answer this open question, a model system was established using the ARD-sensitive apple rootstock M26 (Malus × domestica Borkh. Rosaceae) and GC-MS analysis in combination with an in-house GC-MS database including retention indices. We have detected a total of 35 phytoalexins, i.e. 10 biphenyls and 25 dibenzofurans in root samples, thereby adding eight compounds to the previously reported 27 phytoalexins of Malinae species. When in vitro cultured M26 plantlets were treated with yeast extract, all the 35 phytoalexins were formed in the roots and 85.2% of the total phytoalexin amount was exuded into the culture medium. In roots of M26 plants grown in ARD soil in pot, 26 phytoalexins were detected and their exudation was demonstrated using two independent approaches of collecting root exudates. In a modified dipping experiment and a soil-hydroponic hybrid setup, the exudation rate was 39.5% and 20.6%, respectively. The exudation rates for individual phytoalexins differed, indicating controlled exudation processes. The exuded phytoalexins may play an important role in shaping the soil microbiome, which appears to greatly influence the development and severity of ARD.

Induction of Phytoalexins Gliceoline and Proteins Related to Defense in Soybean Cotyledon Treated With Yeast

Yeasts can induce mechanisms of plant resistance due to compounds with eliciting characteristics, so the aim of this work was to evaluate the effect of yeast on the induction of phytoalexins gliceoline, peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase from soy cotyledons. To determine the defense enzymes, soybean seeds were sown and the cotyledons treated with sterile distilled water, Cryptococcus laurentii (AH 03-1), Pichia guilliermondii (AH 16-2), Rhodotorula glutinis (AH 14-3), Sporidiobolus johnsonii (AH 16-1) and Zygoascus hellenicus (AH 14-1). Biochemical analyzes of the formation of phytoalexins and the activity of the enzymes peroxidase, polyphenoloxidase, phenylalanine ammonia-lyase and total proteins were performed. For phytoalexins glycerolins the yeasts Cryptococcus laurentii (AH 03-1) and Zygoascus hellenicus (AH 14-1) promoted an increase of 83.65% and 78.75% in the formation of this compound. Cryptococcus laurentii (AH 03-1) increased peroxidase activity by 36.84%, while for polyphenoloxidase, the Pichiaguilliermondii e yeasts (AH 16-2), Rhodotorula glutinis (AH 14-3), Sporidiobolus johnsonii (AH 16-1) and Zygoascus hellenicus (AH 14-1), increased the activity of this enzyme by 33.33%, 28.00%, 33.33% and 33.33%, respectively. For phenylalanine ammonia-lyase, Cryptococcus laurentii (AH 03-1) and Zygoascushellenicus (AH 14-1) promoted an increase of 75.57% and 78.86%, respectively, in their activity. The results demonstrate the potential of yeasts studied in the induction of phytoalexins glyceolins and in the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase in soybean cotyledons.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus×domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M.domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V.inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

CYP71Z18 overexpression confers elevated blast resistance in transgenic rice.

CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.

English

The objective of this work was to evaluate the population variation dynamics of Candidatus Liberibacter spp. under phosphite applications. The 'Ponkan' tangerines (Citrus reticulata Blanco cv. Ponkan) with symptoms and a high degree of huanglongbing (HLB) severity were used for the test. 'Ponkan' tangerine plants were treated with potassium phosphite leaf applications at a dose of 2.5 L ha-1. Applications frequency was intervals of 25 days with a total of two applications. There was variation in bacterium Candidatus Liberibacter spp. population, depending on the application of phosphite. Phosphites have an indirect action on pathogens control, stimulating the formation of phytoalexins, a natural self-defense substance of plants. Indirect effects of these products were reported in citrus seedlings presenting symptoms of phosphorus deficiency, which when treated with foliar applications of potassium phosphite overcame these symptoms, restoring plant growth. Candidatus Liberibacter spp. population in 'Ponkan' (Citrus reticulata Blanco cv. Ponkan) leaves was reduced and the phosphorus and potassium contents increased with 2.5 L ha-1 of potassium phosphite by means of leaf application. Key words: Citrus reticulata, leaf nutrition, huanglongbing, phosphorus, potassium.

Transcriptional response of a novel HpCDPK1 kinase gene from Hippeastrum x hybr. to wounding and fungal infection

Calcium dependent protein kinases (CDPK) are well established plant sensor and effectors for calcium ions and participate in regulation of multiple abiotic and biotic stress responses in plant cells. Here we present the identification and characterization of a new CDPK kinase gene from bulbous plant Hippeastrum x hybr. and examine the role of this kinase in stress responses leading to phytoalexin (PA) production in plant tissues. In the previous research, it was shown that Hippeastrum bulbs mechanically wounded or infected with Peyronellaea curtisii (=Phoma narcissi) are inducted to an antifungal red substance synthesis. In this research, we demonstrated Ca2+ dependence of the phytoalexin production by wounded bulbs. Furthermore, the isolated HpCDPK1 cDNA for ORF was found to be 1596bp long and encoded 531 amino acid protein with CDPK kinase activity, as was shown by recombinant GST-HpCDPK1 enzyme production and analysis. HpCDPK1 transcript was present in all vegetative and chosen generative organs of Hippeastrum plant. The dynamics of the observed HpCDPK1 mRNA changes in bulbs depended on stressor type. The mechanical injury caused one wave of transcript increase while more complex transcript changes were observed within 48h after Peyronellaea inoculation. In plant bulbs already accumulating red phytoalexin, increases in HpCDPK1 mRNA level were observed at certain intervals within 48h whereas, in the case of fungal infection, only one big increment in the transcript amount at the 10th minute after inoculation was detected. The observed transcriptional response of HpCDPK1 gene to wounding and pathogen infection stress suggests a positive correlation with phytoalexin synthesis and maintenance in bulb tissues and puts more light on CDPK kinase role in the plant stress response regulation. This also bears some potential for understanding the mechanism of a phytoalexin formation.

BIPHENYLS AND DIBENZOFURANS - FIRE BLIGHT-INDUCED PHYTOALEXINS OF PEAR

After fire blight inoculation, shoots of apple and pear formed biphenyls and dibenzofurans as defense compounds. The phytoalexins were only present in the transition zone of stems, whereas leaves were devoid of these compounds. In shoots of the apple cultivar ‘Holsteiner Cox’, four biphenyls and two dibenzofurans accumulated in the downward advancing transition zone six weeks after Erwinia amylovora inoculation. In the pear cultivar ‘Conference’, three biphenyls and one dibenzofuran were detected. The total phytoalexin content in the transition zone of pear was much lower than that in apple. In addition to ‘Conference’, two other pear cultivars, ‘Alexander Lucas’ and ‘Harrow Sweet’ were investigated for their phytoalexin formation in response to fire blight inoculation. Differences in the morphological changes as well as in the biphenyl and dibenzofuran concentration and pattern were observed between these three genotypes. 'Harrow Sweet', classified as most resistant to fire blight, showed the highest phytoalexin concentration when compared to ‘Alexander Lucas’ and ‘Conference’ and its transition zone stopped the downward migration 12 days after inoculation. Five biphenyls and one dibenzofuran were detectable in this pear cultivar.

Elicitor activity of chitosan and arachidonic acid: Their similarity and distinction

Two elicitors-chitosan and arachidonic acid-induced the same defense responses in potatoes, stimulating the processes of wound reparation and inducing the formation of phytoalexins, inhibitors of proteinase, and active forms of oxygen. However, chitosan induced the defense potential of plant tissues at concentrations higher than those of arachidonic acid. The protective action of chitosan was defined by two parameters, i.e., the ability to induce the immune responses in plant tissues and to exhibit a toxic effect on the pathogen development, causing late blight and seedling blight, whereas the elicitor effect of arachidonic acid depended on its ability to induce the defense potential of plant tissues only.

Iron availability alters ascorbate-induced stress metabolism in Glehnia littoralis root cultures

Our previous study indicated that formation of furanocoumarin phytoalexins could be induced in Glehnia littoralis root cultures by treatment with 10–40mM ascorbic acid (AsA). This furanocoumarin production is much less evident when G. littoralis roots are treated with AsA under iron-deficient conditions. Instead, two large unknown peaks appeared in the HPLC chromatogram, whose chemical structures were elucidated by spectroscopic methods as being 6, β-dihydroxyphenethyl ferulate (DF) and 6-hydroxyphenethyl ferulate (HF), respectively. Their maximal level of induction was observed at 20mM AsA, and the production of DF always exceeded that of HF. This is the first report of these compounds in G. littoralis and of the modulation of the phytoalexin biosynthetic pathway in G. littoralis by iron deficiency.

Increasing Soy Isoflavonoid Content and Diversity by Simultaneous Malting and Challenging by a Fungus to Modulate Estrogenicity

Soybeans were germinated on a kilogram-scale, by the application of malting technology used in the brewing industry, and concomitantly challenged with Rhizopus microsporus var. oryzae. In a time-course experiment, samples were taken every 24 h for 10 days, and the isoflavonoid profile was analyzed by RP-UHPLC-MS. Upon induction with R. microsporus, the isoflavonoid composition changed drastically with the formation of phytoalexins belonging to the subclasses of the pterocarpans and coumestans and by prenylation of the various isoflavonoids. The pterocarpan content stabilized at 2.24 mg of daidzein equivalents (DE) per g after ∼9 days. The levels of the less common glyceofuran, glyceollin IV, and V/VI ranged from 0.18 to 0.35 mg DE/g and were comparable to those of the more commonly reported glyceollins I, II, and III (0.22-0.32 mg DE/g) and glycinol (0.42 mg DE/g). The content of prenylated isoflavones after the induction process was 0.30 mg DE/g. The total isoflavonoid content increased by a factor of 10-12 on DW basis after 9 days, which was suggested to be ascribable to de novo synthesis. These changes were accompanied by a gradual increase in agonistic activity of the extracts toward both the estrogen receptor α (ERα) and ERβ during the 10-day induction, with a more pronounced activity toward ERβ. Thus, the induction process yielded a completely different spectrum of isoflavonoids, with a much higher bioactivity toward the estrogen receptors. This, together with the over 10-fold increase in potential bioactives, offers promising perspectives for producing more, novel, and higher potency nutraceuticals by malting under stressed conditions.

Localized Production of Phytoalexins by Peanut (Arachis hypogaea) Kernels in Response to Invasion by Aspergillus Species

Peanuts respond to fungal infection by synthesizing phytoalexins, most of which are antibiotic stilbenes. The mechanism and dynamics of phytoalexin formation in the peanut have not been studied. One of the most popular peanut cultivars in the southeastern United States, Georgia Green, was investigated for its ability to produce phytoalexins in response to infection by soil fungal strains. The experimental design allowed for study of phytoalexin production in peanut kernels layer-by-layer. The layers were dissected from different depths of the kernel starting from the infected area down to healthier tissues. Six peanut phytoalexins, trans-resveratrol, trans-arachidin-1, trans-arachidin-2, trans-arachidin-3, trans-3-isopentadienyl-4,3',5'-trihydroxystilbene, and SB-1, were detected in the kernel slices and quantitated. All of the fungal strains triggered phytoalexin production; however, the composition of phytoalexins varied significantly by layer. After incubation for 24 h, tissues remote from the infected area produced almost exclusively trans-resveratrol, whereas closer to the infected area tissues synthesized all six phytoalexins. In all of the experiments, after 48 h of fungal growth, deeper layers produced all tested phytoalexins. There was a significant difference in phytoalexin production elicited by some fungal isolates. No association was observed between phytoalexin production and toxigenic potential of fungal strains that elicited the production in mature peanut kernels.

Regulation of potato immune responses by laminarin

Laminarin blocks potato immune responses by inhibiting the reaction of oversensitivity, formation of phytoalexins, wound repair, and the activity of proteinase inhibitors. It was found that laminarin exhibits antielicitor activity. Addition of salicylic acid to laminarin enhances its immunosuppressing effect, which becomes systemic.

Release of phytoalexins and related isoflavonoids from intact chickpea seedlings elicited with reduced glutathione at root level

Induction of phytoalexin formation in roots of chickpea ( Cicer arietinum L.) seedlings treated with the reduced form of glutathione (GSH) was investigated. Accumulation of phytoalexins and constitutive isoflavonoids upon GSH treatment was assessed by high performance liquid chromatography analysis of extracts from roots and liquid incubating media of seedlings, whereas the requirement for concurrent de novo synthesis of induced compounds was assessed in elicitation studies conducted in the presence of key enzyme inhibitors of the phenylpropanoid pathway and/or 〚U- 14C〛-L-phenylalanine. The results indicate that: (a) GSH elicits the formation not only of the pterocarpan phytoalexins medicarpin and maackiain, but also that of the constitutive isoflavones biochanin A and formononetin and, in seedlings older than 4 d, that of the isoflavanones homoferreirin and cicerin. (b) GSH-induced secondary metabolites do not accumulate in plant root tissue; rather, they are released into the surrounding external medium. (c) Phytoalexins and isoflavonoids induced by GSH are de novo synthesized from early phenylpropanoid pathway precursors rather than derived from pre-existing conjugate forms or immediate precursors. (d) Because of metabolic competence and/or proximity, only a small proportion of root cells is responsive to GSH elicitation, hence the very small amounts or absence of induced compounds detected in root tissue samples.

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi.

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H2O2 in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H2O2 production started after a lag time of 30-40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H2O2 did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H2O2 (0.1-1 mM). These results suggest that H2O2 production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H2O2 is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.

Receptor-mediated MAP kinase activation in plant defense.

Plants mount a complex array of defense reactions in response to attack by pathogens. Initiation of these events depends on perception and signal transduction of elicitors, which are plant-derived or pathogen-derived signals, that give rise to transcriptional activation of defense-related genes as well as to changes in activities of enzymes involved in cell wall reinforcement and oxygen radical formation. An oligopeptide, identified within a 42 kDa glycoprotein elicitor from Phythophthora sojae, activates in parsley cells typical plant defense reactions, enabling researchers to study plant-pathogen interaction at the single cell level. The oligopeptide elicitor was found to be necessary and sufficient to stimulate a complex defense response in parsley cells, comprising H+/Ca2+ influxes, K+/Cl- effluxes, activation of a mitogen-activated protein (MAP) kinase, an oxidative burst, defense-related gene activation, and phytoalexin formation.

Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen

Methyl viologen (MV) (20–150 μM), a generator of superoxide anion (O2−), but not hydrogen peroxide (H2O2) (10 μM–2 mM) triggered the formation of cryptotanshinone (a phytoalexin) in cultures of both crown galls and hairy roots of Salvia miltiorrhiza. MV also inhibited the biomass formation and decreased the contents of phenolic acids in both cultures whereas H2O2 did not. In addition, MV and yeast elicitor induced cryptotanshinone formation synergistically only in crown gall cultures. Treatment of the cultures with 3.3 μM diphenylene iodonium, an inhibitor of NAD(P)H oxidase, did not exhibit any detrimental effect on the yeast elicitor-induced cryptotanshinone formation in hairy root cultures whereas 1 μM diphenylene iodonium was inhibitory on yeast elicitor-induced cryptotanshinone formation in crown gall cultures.

566 Induction of Defenses Against Pathogens of Fruit and Vegetables by Elicitors

Fruit and vegetables are highly perishable and postharvest pathogens are one of the major causes in the early termination of their shelf life. Although synthetic fungicide are effective against postharvest pathogens, they face imminent problems. One alternate approach is the induction of host resistance against postharvest diseases. We tested the efficacy of chitosan, Milsana®, and Bion® on carrot, tomato, potato, green pepper, and strawberries against postharvest fungal and bacterial rots. Chitosan was not only antimicrobial but also induced resistance against postharvest infections of Alternaria, Botrytis, and Erwinia in tomato, green pepper, carrot, and potato. Preharvest sprays of chitosan induced resistance against postharvest infection of Botrytis cinerea in strawberries and improved the storage quality. Ultrastructure and cytochemical investigations showed that chitosan not only induced several morphological alterations in B. cinerea and Alternaria alternata inhibiting the production of pathogenic factors by the fungi, but also stimulated various host defense reactions, such as papilla formation and induction of defense enzymes, phytoalexins, and phenols. Application of Milsana®, a plant extract, and Bion®, an activator of host defenses, protected carrots from postharvest decay by B. cinerea. Milsana® was effective at 4 °C, while Bion® was effective at both 4 and 13 °C storage temperatures. Thus, there is great potential for defense inducers in postharvest disease control of fruit and vegetables.