Biological Control of Citrus Postharvest Phytopathogens.
Moraes Bazioli Jaqueline,Belinato João Raul,Costa Jonas Henrique,Akiyama Daniel Yuri,Pontes João Guilherme de Moraes,Kupper Katia Cristina,Augusto Fabio,de Carvalho João Ernesto,Fill Taícia Pacheco
Citrus are vulnerable to the postharvest decay caused by , , and , which are responsible for the green mold, blue mold, and sour rot post-harvest disease, respectively. The widespread economic losses in citriculture caused by these phytopathogens are minimized with the use of synthetic fungicides such as imazalil, thiabendazole, pyrimethanil, and fludioxonil, which are mainly employed as control agents and may have harmful effects on human health and environment. To date, numerous non-chemical postharvest treatments have been investigated for the control of these pathogens. Several studies demonstrated that biological control using microbial antagonists and natural products can be effective in controlling postharvest diseases in citrus, as well as the most used commercial fungicides. Therefore, microbial agents represent a considerably safer and low toxicity alternative to synthetic fungicides. In the present review, these biological control strategies as alternative to the chemical fungicides are summarized here and new challenges regarding the development of shelf-stable formulated biocontrol products are also discussed.
Effects of dog rose and watercress extracts on control of green mould decay and postharvest quality of orange fruits.
Jafari Sajad,Hassandokht Mohammadreza,Javan-Nikkhah Mohammad
Natural product research
Minimum inhibitory concentration (MIC) and IC50 values and total phenolics of dog rose fruits were 72.5-80 μL mL(- 1), 130 μg mL(- 1) and 5.7 mg GA g(- 1), respectively. The Fashand watercress population and dog rose extracts exhibited mycelia growth inhibition values of 45.08% and 37.12%, respectively. The results of in vivo study indicated that the treatment of inoculated fruits with both methanol extracts especially the watercress plant extract considerably controls the citrus fruits decay (Penicillium digitatum) up to 73%. In conclusion, methanol extracts of dog rose fruits and especially watercress plant had the potential to be used against citrus green mould and even for the improvement of postharvest quality of orange fruits.
Control of Citrus Post-harvest Green Molds, Blue Molds, and Sour Rot by the Cecropin A-Melittin Hybrid Peptide BP21.
Wang Wenjun,Liu Sha,Deng Lili,Ming Jian,Yao Shixiang,Zeng Kaifang
Frontiers in microbiology
In this study, the activity of the cecropin A-melittin hybrid peptide BP21 (Ac-FKLFKKILKVL-NH) in controlling of citrus post-harvest green and blue molds and sour rot and its involved mechanism was studied. The minimum inhibitory concentrations of BP21 against , , and were 8, 8, and 4 μmol L, respectively. BP21 could inhibit the growth of mycelia, the scanning electron microscopy results clearly showed that the mycelia treated with BP21 shrank, formed a rough surface, became distorted and collapsed. Fluorescent staining with SYTOX Green (SG) indicated that BP21 could disintegrate membranes. Membrane permeability parameters, including extracellular conductivity, the leakage of potassium ions, and the release of cellular constituents, visibly increased as the BP21 concentration increased. Gross and irreversible damage to the cytoplasm and membranes was observed. There was a positive correlation between hemolytic activity and the concentration of BP21. These results suggest peptide BP21 could be used to control citrus post-harvest diseases.
Targeted transcriptional and proteomic studies explicate specific roles of Bacillus subtilis iturin A, fengycin, and surfactin on elicitation of defensive systems in mandarin fruit during stress.
Tunsagool Paiboon,Leelasuphakul Wichitra,Jaresitthikunchai Janthima,Phaonakrop Narumon,Roytrakul Sittiruk,Jutidamrongphan Warangkana
Application of Bacillus cyclic lipopeptides (CLPs); fengycin, iturin A and surfactin has shown a great potential in controlling the spread of green mold pathogen invasion (Penicillium digitatum) in wounded mandarin fruit during postharvest period. The limited defensive protein profiles followed specific expression of pivotal genes relating to plant hormone mediating signaling pathways of the CLPs' action on stimulating host plant resistance have been exhibited. The present study aimed to elucidate the specific effect of individual CLP obtained from Bacillus subtilis ABS-S14 as elicitor role on activation of plant defensive system at transcriptional and proteomic levels with and without P. digitatum co-application in mandarin fruit. Fengycin and iturin A elevated the gene expression of PAL, ACS1, ACO, CHI, and GLU while significantly stimulating plant POD transcription was only detected in the treatments of surfactin both with and without following P. digitatum. An increase of LOX and PR1 gene transcripts was determined in the treatments of individual CLP with fungal pathogen co-application. Fengycin activated production of unique defensive proteins such as protein involved in ubiquinone biosynthetic process in treated flavedo without P. digitatum infection. Proteins involved in the auxin modulating pathway were present in the iturin A and surfactin treatments. CLP-protein binding assay following proteome analysis reveals that iturin A attached to 12-oxophytodienoate reductase 2 involved in the oxylipin biosynthetic process required for jasmonic acid production which is implicated in induced systemic resistance (ISR). This study suggests specific elicitor action of individual CLP, particularly iturin A showed the most powerful in stimulating the ISR system in response to stresses in postharvest mandarins.
Inhibition of Key Citrus Postharvest Fungal Strains by Plant Extracts In Vitro and In Vivo: A Review.
Chen Jinyin,Shen Yuting,Chen Chuying,Wan Chunpeng
Plants (Basel, Switzerland)
fruits are subjected to a diversity of postharvest diseases caused by various pathogens during picking, packing, storage and transportation. Green and blue molds, caused by and , respectively, are two major postharvest citrus diseases and cause significant economic losses during the commercialization phase. Currently, the control of postharvest citrus diseases relies mainly on the use of synthetic fungicides, which usually result in the resistance against fungal attack, environment pollution and health hazards. In recent years, much attention has been given to the preservation of citrus by naturally isolated edible plant extracts, medicinal plant extracts, extracts and volatiles, et al. Scientists worldwide devote their time and energy to discover the high effect, low toxicity, safety and inexpensive plant-derived fungicides. The current review will highlight plant-derived fungicides and chemical constituents that aim to inhibit and in vitro and in vivo. Coatings enriched with plant extracts could be good alternative methods for fruits preservation. Problems and prospects of the research and development of plant-derived natural fungicides will also be discussed in this article.
Novel synthesized 2, 4-DAPG analogues: antifungal activity, mechanism and toxicology.
Gong Liang,Tan Haibo,Chen Feng,Li Taotao,Zhu Jianyu,Jian Qijie,Yuan Debao,Xu Liangxiong,Hu Wenzhong,Jiang Yueming,Duan Xuewu
2, 4-Diacetylphloroglucinol (2,4-DAPG), a natural phenolic compound, has been investigated in light of its biological activities against plant pathogens. To improve its potential application, fourteen 2,4-DAPG analogous were synthesized through the Friedel-Crafts reaction using acyl chlorides and phloroglucinol. Of the 2,4-DAPG derivatives, MP4 exhibited much higher antifungal activity against Penicillium digitatum and P. italicum, the major pathogenic fungi in citrus fruit, than 2, 4-DAPG in vitro, and significantly inhibited the development of decay in harvested mandarin (Citrus reticulata Blanco cv. Shatang.) fruit in vivo. It was found that MP4 resulted in the wrinkle of the hyphae in both fungi with serious folds and breakage. In addition, the expression of several cytochrome P450 (CYP) genes were also modified in both fungi by MP4, which might be associated with the disorder of cell membrane formation. Furthermore, the toxicology of MP4 by evaluating the cell proliferation effect on human normal lung epithelial (16HBE) and kidney 293 (HEK293) cells, was significantly lower than that of albesilate, a widely used fungicide in harvested citrus fruit. In summary, the synthesized MP4 has shown a great potential as a novel fungicide that might be useful for control of postharvest decay in citrus fruit.
Mechanisms of action for 2-phenylethanol isolated from Kloeckera apiculata in control of Penicillium molds of citrus fruits.
Liu Pu,Cheng Yunjiang,Yang Meng,Liu Yujia,Chen Kai,Long Chao-An,Deng Xiuxin
BACKGROUND:Green and blue mold decay, caused by Penicillium digitatum and P. italicum, respectively, are important postharvest diseases of citrus. Biocontrol by microbes is an alternative to synthetic fungicide application. In this study, the antagonistic yeast strain Kloeckera apiculata 34-9 was used to investigate the action mechanisms involved in the biocontrol of postharvest diseases. RESULTS:An antifungal substance, 2-phenylethanol (PEA), was isolated from K. apiculata and demonstrated to have antimicrobial activity against selected phytopathogenic fungi. Experiments on P. italicum cells identified the mitochondria and the nucleus as particularly sensitive to inhibition. Regulation of P. italicum gene expression was investigated using RNA-Seq. PEA up-regulated genes involved with the peroxisome, regulation of autophagy, phosphatidylinositol signaling system, protein processing in endoplasmic reticulum, fatty acid metabolism, and inhibited ribosome, RNA polymerase, DNA replication, amino acid biosynthesis, aminoacyl-tRNA biosynthesis and cell cycle. Inhibitory responses revealed by RNA-Seq suggest that PEA might compete for attachment on the active site of phenylalanyl-tRNA synthetase (PheRS). CONCLUSION:This study provided new insight on the mode of action of biocontrol yeast agents in controlling postharvest pathogenic fungi.
Potential of a new strain of Bacillus amyloliquefaciens BUZ-14 as a biocontrol agent of postharvest fruit diseases.
Calvo H,Marco P,Blanco D,Oria R,Venturini M E
The biocontrol potential of the Bacillus amyloliquefaciens strain BUZ-14 was tested against the main postharvest diseases of orange, apple, grape and stone fruit. After characterizing the temperature and pH growth curves of strain BUZ-14, its in vitro antifungal activity was determined against Botrytis cinerea, Monilinia fructicola, M. laxa, Penicillium digitatum, P. expansum and P. italicum. Subsequently, in vivo activity was tested against these pathogens by treating fruit with cells, endospores and cell-free supernatants. The in vitro results showed that BUZ-14 inhibited the growth of all the pathogens tested corresponding to the least susceptible species, P. italicum, and the most susceptible, M. laxa. In vivo tests corroborated these results as most of the treatments decreased the incidence of brown rot in stone fruit from 100% to 0%, establishing 10 CFU mL as the minimum inhibitory concentration. For the Penicillium species a preventive treatment inhibited P. digitatum and P. italicum growth in oranges and reduced P. expansum incidence in apples from 100% to 20%. Finally, it has been demonstrated that BUZ-14 was able to survive and to control brown rot in peaches stored at cool temperatures, making it a very suitable biocontrol agent for application during the post-harvest storage and marketing of horticultural products.
Effect of applying cinnamaldehyde incorporated in wax on green mould decay in citrus fruits.
Duan Xiaofang,OuYang Qiuli,Tao Nengguo
Journal of the science of food and agriculture
BACKGROUND:Green mould caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Cinnamaldehyde (CA) is a food additive that has potential use in controlling postharvest disease of fruits and vegetables. In this study, the effectiveness of wax with CA (WCA) in controlling Ponkan (Citrus reticulata Blanco) green mould was investigated. RESULTS:The mycelial growth of P. digitatum was inhibited by CA in a dose-dependent manner. The minimum inhibitory concentration and minimum fungicidal concentration (MFC) were both 0.50 mL L . In vivo tests demonstrated that WCA (1 × and 10 × MFC) applied to Ponkan fruits inoculated with P. digitatum could significantly decrease the incidence of green mould for up to 5 days. The WCA treatment increased the activities of catalase, superoxide dismutase, peroxidase, phenylalanine ammonia lyase, polyphenol oxidase, as well as the total phenols and flavonoids contents. Meanwhile, the treatment remarkably decreased the weight loss rate of fruits and maintained fruit quality. These results indicated that WCA treatment might induce defence responses against green mould in citrus fruit. CONCLUSION:Our findings suggest that WCA might be a promising approach in controlling green mould of citrus fruits. © 2017 Society of Chemical Industry.
Control of citrus molds using bioactive coatings incorporated with fungal chitosan/plant extracts composite.
Tayel Ahmed A,Moussa Shaaban H,Salem Mohammed F,Mazrou Khaled E,El-Tras Wael F
Journal of the science of food and agriculture
BACKGROUND:The ongoing postharvest loss in citrus fruits, due to fungal infection, is a chronic economic and agricultural problem. Most of citrus damage is caused by Penicillium spp., e.g. green mold (P. digitatum) and blue mold (P. italicum). Fungal chitosan, from Mucor rouxii, and plant extracts from cress seeds, olive leaves, pomegranate peels and senna pods, were evaluated as antifungal agents against the phytopathogenic fungi, P. digitatum and P. italicum, using in vitro qualitative and quantitative assays. RESULTS:All natural agents tested exhibited potent antifungal activity; the most powerful agent was cress (Lepidium sativum) seed extract, followed by pomegranate (Punica granatum) peel extract. Fungal chitosan also had a remarkable fungicidal potentiality using both evaluation assays. Penicillium digitatum was generally more resistant than P. italicum toward all examined agents. The incorporation of each individual natural agent in coating material resulted in a great reduction in fungal growth and viability. The addition of chitosan combined with cress and pomegranate extracts, to the coating materials, prevented coated citrus fruit from decay by green and blue mold for a 2-week storage period. CONCLUSION:Natural derivatives could be recommended as powerful antifungal alternatives to protect citrus fruits from postharvest fungal decay.
Evaluation of the activity of the antifungal PgAFP protein and its producer mould against Penicillium spp postharvest pathogens of citrus and pome fruits.
Delgado Josué,Ballester Ana-Rosa,Núñez Félix,González-Candelas Luis
Postharvest fungal diseases are among the main causes of fresh fruit losses. Chemical control is against claims for "natural" or "chemical-free" products. Biocontrol agents, such as antifungal proteins or their producing moulds, may serve to combat unwanted pathogens. Since the effectiveness of these bioprotective agents depends on the food substrate, their effect must be tested on fruits. The objective of this work was to study the effect of the antifungal protein PgAFP and its producer, Penicillium chrysogenum, against Penicillium expansum and Penicillium digitatum growth on apple and oranges respectively, and the PgAFP effect on eleven P. expansum, Penicillium italicum, and P. digitatum strains in vitro, and on patulin production on apple substrate. The sensitivity upon PgAFP was P. digitatum > P. expansum > P. italicum. In oranges, broadly, no inhibitory effect was obtained. PgAFP and P. chrysogenum did not inhibit the P. expansum CMP-1 growth on Golden Delicious apples, however, a successful effect was achieved on Royal Gala apples. On apple substrate, patulin production by P. expansum CMP-1 rose in parallel to PgAFP concentrations, linked with high reactive oxygen species levels. PgAFP cannot be proposed as a bioprotective agent on apple. However, P. chrysogenum is a promising agent to be used on Royal Gala apples.
Antagonistic Activity and the Mechanism of Bacillus amyloliquefaciens DH-4 Against Citrus Green Mold.
Chen Kai,Tian Zhonghuan,Luo Yuan,Cheng Yunjiang,Long Chao-An
Citrus fruit usually suffer significant losses during the storage and transportation stages. Green mold, a postharvest rot of citrus fruit caused by Penicillium digitatum, is one of the most serious fungal diseases. In this study, the antagonist strain DH-4 was identified as Bacillus amyloliquefaciens according to morphological observation and 16S ribosomal DNA analysis. In addition, it showed broad antifungal activity, especially the suppression of Penicillium spp. The culture filtrate of strain DH-4 exhibited apparent activity against P. digitatum in vitro and in vivo. In storage, the culture filtrate with DH-4 in it showed a better antiseptic effect. The antifungal substances in the culture filtrate, produced by strain DH-4, displayed stable activity in various extreme conditions. In addition, the antifungal substances in the culture filtrate were identified as macrolactin, bacillaene, iturins, fengycin, and surfactin by ultraperformance liquid chromatography (UPLC) electrospray ionization mass spectrometry analysis. The UPLC fractions containing these antifungal compounds were basically heat tolerant and all responsible for the antagonistic activity against P. digitatum. Transmission electron microscope observation indicated that the antifungal substances might cause abnormalities in the P. digitatum cellular ultrastructure, which could be the possible mode of action of B. amyloliquefaciens against P. digitatum. In addition, it was confirmed via scanning electron microscope analysis that the main way it inhibited P. digitatum was by secreting antimicrobial compounds without direct interaction. This study contributes to the understanding of the mechanism of B. amyloliquefaciens against citrus green mold as well as providing a potential application for the biocontrol of postharvest rot diseases in citrus fruit.
Mechanisms of N-lauroyl arginate ethyl ester against and subsp. .
Xu Xiao-Hui,Jiang Zeng-Liang,Feng Feng-Qin,Lu Rong-Rong
Journal of food science and technology
The purpose of this study was to investigate the antimicrobial activity and mechanisms of N-lauroyl arginate ethyl ester (LAE) against and subsp. . The minim inhibitory concentrations of LAE against and were found to be 400 and 25 μg/ml, respectively. Loss of intracellular protein and nucleic acid increased significantly, and membrane permeability reached 76.28, 54.29 and 85.20%, respectively, when 400 μg/ml of LAE was applied to the hyphae and spores of and to . Flow cytometry showed that LAE reduced the membrane potential, and the depolarization ratios of and were 98.19 and 97.25% (< 0.05), respectively. Transmission electron microscopy photos revealed that LAE caused a rough surface, irregular cellular organelles, protoplast shrinkage, intracytoplasmic coagulation and empty cavities in all three cell types. These results showed that LAE had notable ability to damage the structure of fungal and bacterial cells, making it a possible alternative chemical for use in the preservation of fruits and vegetables.
Penicillium digitatum infection mechanisms in citrus: What do we know so far?
Costa Jonas Henrique,Bazioli Jaqueline Moraes,de Moraes Pontes João Guilherme,Fill Taícia Pacheco
Penicillium digitatum is the major source of postharvest decay in citrus fruits worldwide. This fungus shows a limited host range, being able to infect mainly mature fruit belonging to the Rutaceae family. This highly specific host interaction has attracted the interest of the scientific community. Researchers have investigated the chemical interactions and specialized virulence strategies that facilitate this fungus's fruit colonization, thereby leading to a successful citrus infection. There are several factors that mediate and affect the interaction between P. digitatum and its host citrus, including hydrogen peroxide modulation, secretion of organic acids and consequently pH control, and other strategies described here. The recently achieved sequencing of the complete P. digitatum genome opened up new possibilities for exploration of the virulence factors related to the host-pathogen interaction. Through such techniques as RNAseq, RT-PCR and targeted gene knockout mediated by Agrobacterium tumefaciens, important genes involved in the fungal infection process in citrus have been reported, helping to elucidate the molecular mechanisms, metabolites and genetic components that are involved in the pathogenicity of P. digitatum. Understanding the infection process and fungal strategies represents an important step in developing ways to protect citrus from P. digitatum infection, possibly leading to more productive citriculture.
Plasma membrane damage contributes to antifungal activity of citronellal against .
Wu Yalan,OuYang Qiuli,Tao Nengguo
Journal of food science and technology
The antifungal activity of citronellal, a typical terpenoid of plant essential oils, against and the possible action mode involved were investigated. Results showed that the mycelial growth and spores' germination of were inhibited by citronellal in a dose-dependent manner. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined to be 1.60 µL/mL and 3.20 µL/mL, respectively. It was found that the plasma membrane of citronellal-treated spores was damaged, as confirmed by the propidium iodide stain results, as well as a higher extracellular conductivity and release of cell constituents in citronellal-treated samples than those of control samples. Moreover, in vivo test results demonstrated that wax + citronellal (WC; 10 × MFC) treatment effectively reduced the incidence of green mold after 5 days of storage at 25 ± 2 °C. These findings suggested that the plasma damage mechanism contributed to the antifungal activity of citronellal against . In addition, citronellal was suggested to be a potential alternative to fungicidal agents in controlling green mold of citrus fruit.
A Damaged Oxidative Phosphorylation Mechanism Is Involved in the Antifungal Activity of Citral against .
OuYang Qiuli,Tao Nengguo,Zhang Miaoling
Frontiers in microbiology
Citral exhibits strong antifungal activity against . In this study, 41 over-expressed and 84 repressed proteins in after 1.0 μL/mL of citral exposure for 30 min were identified by the iTRAQ technique. The proteins were closely related with oxidative phosphorylation, the TCA cycle and RNA transport. The mitochondrial complex I, complex II, complex III, complex IV and complex V, which are involved in oxidative phosphorylation were drastically affected. Among of them, the activities of mitochondrial complex I and complex IV were apparently suppressed, whereas those of mitochondrial complex II, complex III and complex V were significantly induced. Meanwhile, citral apparently triggered a reduction in the intracellular ATP, the mitochondrial membrane potential (MMP) and glutathione content, in contrast to an increase in the glutathione S-transferase activity and the accumulation of reactive oxygen species (ROS). Addition of exogenous cysteine decreased the antifungal activity. In addition, cysteine maintained the basal ROS level, deferred the decrease of MMP and the membrane damage. These results indicate that citral inhibited the growth of by damaging oxidative phosphorylation and cell membranes through the massive accumulation of ROS.
Glucosylceramides are required for mycelial growth and full virulence in Penicillium digitatum.
Zhu Congyi,Wang Mingshuang,Wang Weili,Ruan Ruoxin,Ma Haijie,Mao Cungui,Li Hongye
Biochemical and biophysical research communications
Glucosylceramides (GlcCers) are important lipid components of the membrane systems of eukaryotes. Recent studies have suggested the roles for GlcCers in regulating fungal growth and pathogenesis. In this study, we report the identification and functional characterization of PdGcs1, a gene encoding GlcCer synthase (GCS) essential for the biosynthesis of GlcCers, in Penicilliumdigitatum genome. We demonstrated that the deletion of PdGcs1 in P. digitatum resulted in the complete loss of production of GlcCer (d18:1/18:0 h) and GlcCer (d18:2/18:0 h), a decrease in vegetation growth and sporulation, and a delay in spore germination. The virulence of the PdGcs1 deletion mutant on citrus fruits was also impaired, as evidenced by the delayed occurrence of water soaking lesion and the formation of smaller size of lesion. These results suggest that PdGcs1 is a bona fide GCS that plays an important role in regulating cell growth, differentiation, and virulence of P. digitatum by controlling the biosynthesis of GlcCers.
Influence of α-terpineol on the growth and morphogenesis of Penicillium digitatum.
Jing Guo-Xing,Tao Neng-Guo,Jia Lei,Zhou Hai-En
BACKGROUND:Plant essential oils could act effectively against postharvest diseases, α-terpineol, a typical terpenoid of plant essential oils, exhibited strong antifungal activity in against Penicillium italicum, but the possible action mechanism remains undetermined. In present study, α-terpineol was evaluated for antibacterial activity against Penicillium digitatum along with the mode of their antibacterial action. RESULTS:The results showed that mycelial growth of P. digitatum was strongly inhibited by α-terpineol, with the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 2.00 and 8.00 µl/ml, respectively. Scanning electron microscopy observation revealed that α-terpineol obviously altered the morphology of P. digitatum hyphae by causing the loss of cytoplasm and distortion of mycelia. A rapid increase in the membrane permeability of P. digitatum was observed after treated with MIC or MFC of α-terpineol, evidenced by the release of cell constituents, the extracellular conductivity, and the extracellular pH. In addition, α-terpineol apparently induced a decrease in total lipid contents of P. digitatum cells, indicating the destruction of cell membrane structures after treatment. CONCLUSIONS:Based on our study, α-terpineol might affect the cell wall synthesis and lead to the disruption of cell wall. The cell wall disruption affected fungal morphogenesis, the integrity of membrane and leakage of intracellular components, these results suggested that α-terpineol treatment inhibited the growth of P. digitatum.
Evaluation of Muscodor suthepensis strain CMU-Cib462 as a postharvest biofumigant for tangerine fruit rot caused by Penicillium digitatum.
Suwannarach Nakarin,Bussaban Boonsom,Nuangmek Wipornpan,Pithakpol Wasna,Jirawattanakul Bantoon,Matsui Kenji,Lumyong Saisamorn
Journal of the science of food and agriculture
BACKGROUND:This study investigated both the in vitro and in vivo biofumigant ability of the endophytic fungus Muscodor suthepensis CMU-Cib462 to control Penicillium digitatum, the main cause of tangerine fruit rot. RESULTS:Volatile compounds from M. suthepensis inhibited mycelial growth of the pathogen. The most abundant compound was 2-methylpropanoic acid, followed by 3-methylbutan-1-ol. They showed median effective doses (ED50) on P. digitatum growth of 74.91 ± 0.73 and 250.29 ± 0.29 µL L(-1) airspace respectively. Rye grain was found to be a suitable solid medium for M. suthepensis inoculum production. The results indicated that mycofumigation with a 30 g rye grain culture of M. suthepensis for 12 h controlled tangerine fruit rot. The percentage weight loss and soluble solids concentration of fumigated tangerines were similar to those of non-infected and non-fumigated fruits. CONCLUSION:Muscodor suthepensis has potential as a biofumigant for controlling postharvest disease of tangerine fruit.
Physical properties and antifungal activity of bioactive films containing Wickerhamomyces anomalus killer yeast and their application for preservation of oranges and control of postharvest green mold caused by Penicillium digitatum.
Aloui Hajer,Licciardello Fabio,Khwaldia Khaoula,Hamdi Moktar,Restuccia Cristina
International journal of food microbiology
This study assessed the ability of two bio-based films, obtained from sodium alginate (NaAlg) and locust bean gum (LBG), to protect the viability of Wickerhamomyces anomalus cells and control the growth of Penicillium digitatum. The effect of microbial cell incorporation on physical properties of the developed films was evaluated in terms of barrier, mechanical and optical properties. Furthermore, the application of these two matrices as bioactive coatings was investigated in order to evaluate their efficacy in preserving the postharvest quality of 'Valencia' oranges and inhibiting the growth of P. digitatum on artificially inoculated fruits. Results showed that NaAlg and LBG films were able to maintain more than 85% of the initial W. anomalus yeast population and that the developed films incorporating the killer yeast completely inhibited the growth of P. digitatum in synthetic medium. Likewise, NaAlg and LBG coatings enriched with W. anomalus yeast were effective at reducing weight loss and maintaining firmness of 'Valencia' oranges during storage, and reduced green mold in inoculated fruits by more than 73% after 13 days.
Effect of octanal on the mycelial growth of Penicillium italicum and P. digitatum.
Tao Nengguo,Jia Lei,Zhou Haien,He Xiangli
World journal of microbiology & biotechnology
The present study investigated the antifungal activity of octanal against Penicillium italicum and P. digitatum. Results showed that octanal exhibited strong antifungal activity against the test pathogens in a dose-dependent manner. Scanning electron microscopy observation revealed that octanal obviously altered the morphology of P. italicum and P. digitatum hyphae by causing the loss of cytoplasm and distortion of mycelia. A rapid increase in the membrane permeability of P. italicum and P. digitatum was observed after treated with octanal at minimum inhibitory concentration or minimum fungicidal concentration, evidenced by the release of cell constituents, the extracellular conductivity and the extracellular potential of hydrogen. In addition, octanal apparently induced a decrease in total lipid contents of P. italicum and P. digitatum cells. These results suggested that the antifungal activity of octanal against P. italicum and P. digitatum can be attributed to the disruption of the cell membrane integrity and the leakage of cell components.
Antifungal Activity of α-Sarcin against Penicillium digitatum: Proposal of a New Role for Fungal Ribotoxins.
Citores Lucía,Iglesias Rosario,Ragucci Sara,Di Maro Antimo,Ferreras José M
ACS chemical biology
Among the putative defense proteins that occur in fungi, one of the best studied is α-sarcin, produced by the mold Aspergillus giganteus. This protein is the most significant member of the ribotoxin family, which consists of extracellular rRNA ribonucleases that display cytotoxic activity toward animal cells. Ribotoxins are rRNA endonucleases that catalyze the hydrolysis of the phosphodiester bond between G4325 and A4326 from the rat 28S rRNA. The results of several experimental approaches have led to propose ribotoxins as insecticidal agents. In this work, we report that α-sarcin displays a strong antifungal activity against Penicillium digitatum, being able to enter into the cytosol where it inactivates the ribosomes, thus killing the cells and arresting the growth of the fungus. This is the first time that a ribotoxin has been found to display antifungal activity. Therefore, this protein could play, besides the already proposed insecticidal function, a role in nature as an antifungal agent.
Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mould Penicillium digitatum.
Citores Lucía,Iglesias Rosario,Gay Carolina,Ferreras José Miguel
Molecular plant pathology
The ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter- and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N-glycosylase activity on the sarcin-ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C-terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α-helix and a β-hairpin, similar to the γ-core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.
Effect of NaHCO treatments on the activity of cell-wall-degrading enzymes produced by Penicillium digitatum during the pathogenesis process on grapefruit.
Venditti Tullio,D'hallewin Guy,Ladu Gianfranca,Petretto Giacomo L,Pintore Giorgio,Labavitch John M
Journal of the science of food and agriculture
BACKGROUND:This study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with sodium bicarbonate (NaHCO ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h. RESULTS:Treatment with NaHCO , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, compared with the control or the same treatment at pH 5. In contrast, pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5-treated plugs up to 72 h. CONCLUSION:The rapid increase in pectin methyl esterase activity under alkaline conditions is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact, at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. © 2018 Society of Chemical Industry.
Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC-MS/MS.
Zhang Lu-Lu,Zhang Yan,Ren Jing-Nan,Liu Yan-Long,Li Jia-Jia,Tai Ya-Nan,Yang Shu-Zhen,Pan Si-Yi,Fan Gang
Journal of industrial microbiology & biotechnology
This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.
Anti-fungal activity of Citrus reticulata Blanco essential oil against Penicillium italicum and Penicillium digitatum.
Tao Nengguo,Jia Lei,Zhou Haien
The chemical composition of Citrus reticulata Blanco essential oil was analysed using GC/MS. Monoterpene hydrocarbons (C10H16) constituted the majority (88.96%, w/w) of the total oil. The oils dose-dependently inhibited Penicillium italicum and Penicillium digitatum. The anti-fungal activity of the oils against P. italicum was attributed to citronellol, octanal, citral, decanal, nonanal, β-pinene, linalool, and γ-terpinene, whereas anti-fungal activity against P. digitatum is attributed to octanal, decanal, nonanal, limonene, citral, γ-terpinene, linalool, and α-terpineol. The oils altered the hyphal morphology of P. italicum and P. digitatum by causing loss of cytoplasm and distortion of the mycelia. The oils significantly altered extracellular conductivity, the release of cell constituents, and the total lipid content of P. italicum and P. digitatum. The results suggest that C. reticulata Blanco essential oils generate cytotoxicity in P. italicum and P. digitatum by disrupting cell membrane integrity and causing the leakage of cell components.
Polyhexamethylene guanidine as a fungicide, disinfectant and wound protector in lemons challenged with Penicillium digitatum.
Olmedo Gabriela M,Cerioni Luciana,Sepulveda Milena,Ramallo Jacqueline,Rapisarda Viviana A,Volentini Sabrina I
Citrus green mold, a postharvest disease caused by Penicillium digitatum, provokes important economic losses on lemon production. Here, the effectiveness of polyhexamethylene guanidine (PHMG) to inhibit P. digitatum growth and to control green mold on artificially infected lemons was evaluated. At sublethal concentrations, PHMG inhibited conidia germination and infectivity (5 mg L), and mycelial growth (50 mg L). Viability of conidia was completely suppressed by treatment with 500 mg L PHMG. In this condition, membrane integrity loss, cell wall disruption and ultrastructural alterations were detected, as well as conidia distortion, deformation and collapse. In artificially inoculated lemons, a 30 s-immersion in 500 mg L PHMG completely inhibited green mold. PHMG also exhibited a high disinfectant activity, even in the presence of 1% organic matter, with a better performance than the standard NaClO disinfectant. In addition, 500 mg L PHMG protected wounds against infection. Taken together, our results indicate that PHMG is a promising fungicide for the postharvest control of green mold in lemon packinghouses.
The effect of locust bean gum (LBG)-based edible coatings carrying biocontrol yeasts against Penicillium digitatum and Penicillium italicum causal agents of postharvest decay of mandarin fruit.
Parafati Lucia,Vitale Alessandro,Restuccia Cristina,Cirvilleri Gabriella
Strains belonging to Wickerhamomyces anomalus, Metschnikowia pulcherrima and Aureobasidium pullulans species were tested in vitro as biocontrol agents (BCAs) against the post-harvest pathogenic molds Penicillium digitatum and Penicillium italicum. Moreover, studies aimed at screening the antifungal activity of selected yeast strains in vivo conditions against P. digitatum and P. italicum, and investigated the efficacy of a polysaccharidic matrix, locust bean gum (LBG), enriched with the tested BCAs, in controlling postharvest decays in artificially inoculated mandarins. The population dynamics of BCAs on wounds and the magnitude of peroxidase (POD) and superoxide dismutase (SOD) in fruit tissues were also investigated after treatments of mandarins with antagonistic yeasts. W. anomalus BS91, M. pulcherrima MPR3 and A. pullulans PI1 provided excellent control of postharvest decays caused by P. digitatum and P. italicum on mandarins, both when the yeasts were used alone and in combination with LBG, which enhanced the yeast cell viability over time. Finally, the increased activity of POD and lower decrease in SOD activity in response to BCAs application in mandarin fruits confirmed their involvement in the biocontrol mechanism.
The terpene limonene induced the green mold of citrus fruit through regulation of reactive oxygen species (ROS) homeostasis in Penicillium digitatum spores.
Tao Nengguo,Chen Yue,Wu Yalan,Wang Xiao,Li Lu,Zhu Andan
Herein, the dosage effect of limonene on the P. digitatum spore germination and its regulatory mechanisms were investigated. Results showed that limonene only at low concentrations displayed a stimulatory role, with the optimal concentration being 0.25 μL/mL. GC-MS and GC analysis revealed that limonene contents remained relative stable and no evidence of transformation was observed at stimulatory concentrations. Metabolomics analysis showed that 61 metabolites including organic acids, amino acids, sugars, nucleosides, fatty acids, and their derivatives, were significantly changed (P < 0.05), suggesting the transitions between soluble sugars and energy-related metabolisms. Proteomic analysis demonstrated that proteins in energy-related pathways and ROS homeostasis were also influenced. These were further confirmed by the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST), the contents of reactive oxygen species (ROS), hydrogen peroxide (HO), and glutathione (GSH). Our present research indicates that ROS homeostasis is involved in the limonene induced spore germination of P. digitatum.
Octanal inhibits spore germination of Penicillium digitatum involving membrane peroxidation.
Dou Shiwen,Liu Shengquan,Xu Xiaoyong,OuYang Qiuli,Tao Nengguo
Octanal is a potential alternative to chemical fungicides in controlling postharvest disease of citrus fruit. In this study, the antifungal activity and the underlying mechanism of octanal against spore germination of Penicillium digitatum, one of the main postharvest pathogens in citrus, were investigated. Results showed that octanal at different concentrations (0, 0.25, 0.50, 1.00, 2.00 μl/ml) inhibited the growth of P. digitatum spores in a dose-dependent manner. The morphology and the membrane permeability of P. digitatum spores were visibly altered by 0.25 and 2.00 μl/ml of octanal. Meanwhile, octanal decreased the total lipids contents of P. digitatum spores, indicating that the membrane integrity is damaged. Furthermore, octanal apparently induced the massive accumulation of total malonaldehyde (MDA) and the reactive oxygen species (ROS). An increase in the activities of lipoxygenase (LOX), NADH oxidase, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) was also observed. These results suggested that a membrane damage mechanism involving membrane peroxidation might contribute to the antifungal activity of octanal against P. digitatum spores.
Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.
OuYang Qiuli,Tao Nengguo,Jing Guoxing
BACKGROUND:Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. RESULTS:In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. CONCLUSION:Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.
Edible coatings incorporating pomegranate peel extract and biocontrol yeast to reduce Penicillium digitatum postharvest decay of oranges.
Kharchoufi Samira,Parafati Lucia,Licciardello Fabio,Muratore Giuseppe,Hamdi Mokthar,Cirvilleri Gabriella,Restuccia Cristina
This study investigated the potential use of two edible coatings, chitosan (CH) and locust bean gum (LBG), which incorporated chemically characterized water pomegranate peel extract (WPPE) or methanol pomegranate peel extract (MPPE) and the biocontrol agent (BCA) Wickerhamomyces anomalus, to control the growth of Penicillium digitatum and to reduce the postharvest decay of oranges. CH and LBG including pomegranate peel extracts (PPEs) at different concentrations were tested in vitro against P. digitatum to determine their antifungal efficacy; at the same time, the tolerance of viable cells of W. anomalus to increasing concentrations of WPPE and MPPE extracts was assessed. The potential application of selected bioactive coatings was evaluated in vivo on oranges, which had been artificially inoculated with P. digitatum, causal agent of green mold decay. CH incorporating MPPE or WPPE at all concentrations was able to inhibit in vitro P. digitatum, while LBG was active only at the highest MPPE or WPPE concentrations. W. anomalus BS91 was slightly inhibited only by MPPE-modified coatings, while no inhibition was observed by WPPE, which was therefore selected for the in vivo trials on oranges artificially inoculated with P. digitatum. The experimental results proved that the addition of 0.361 g dry WPPE/mL, both to CH and LBG coatings, significantly reduced disease incidence (DI) by 49 and 28% respectively, with respect to the relative controls. Besides the combination CH or LBG + WPPE, the addition of W. anomalus cells to coatings strengthened the antifungal effect with respect to the relative controls, as demonstrated by the significant reduction of DI (up to 95 and 75% respectively). The findings of the study contribute to the valorization of a value-added industrial byproduct and provide a significant advancement in the development of new food protectant formulations, which benefit from the synergistic effect between biocontrol agents and natural bioactive compounds.
Citral exerts its antifungal activity against Penicillium digitatum by affecting the mitochondrial morphology and function.
Zheng Shiju,Jing Guoxing,Wang Xiao,Ouyang Qiuli,Jia Lei,Tao Nengguo
This work investigated the effect of citral on the mitochondrial morphology and function of Penicillium digitatum. Citral at concentrations of 2.0 or 4.0 μL/mL strongly damaged mitochondria of test pathogen by causing the loss of matrix and increase of irregular mitochondria. The deformation extent of the mitochondria of P. digitatum enhanced with increasing concentrations of citral, as evidenced by a decrease in intracellular ATP content and an increase in extracellular ATP content of P. digitatum cells. Oxygen consumption showed that citral resulted in an inhibition in the tricarboxylic acid cycle (TCA) pathway of P. digitatum cells, induced a decrease in activities of citrate synthetase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinodehydrogenase and the content of citric acid, while enhancing the activity of malic dehydrogenase in P. digitatum cells. Our present results indicated that citral could damage the mitochondrial membrane permeability and disrupt the TCA pathway of P. digitatum.
Detection and classification of citrus green mold caused by Penicillium digitatum using multispectral imaging.
Ghanei Ghooshkhaneh Narges,Golzarian Mahmood Reza,Mamarabadi Mojtaba
Journal of the science of food and agriculture
BACKGROUND:Fungal decay is a prevalent condition that mainly occurs during transportation of products to consumers (from harvest to consumption) and adversely affects postharvest operations and sales of citrus fruit. There are a variety of methods to control pathogenic fungi, including UV-assisted removal of fruit with suspected infection before storage, which is a time-consuming task and associated with human health risks. Therefore it is essential to adopt efficient and dependable alternatives for early decay detection. In this study, detection of orange decay caused by Penicillium genus fungi was examined using spectral imaging, a novel automated inspection technique for agricultural products. RESULTS:The reflectance parameter (including mean reflectance) and reflectance distribution parameters (including standard deviation and skewness) of surfaces were extracted from decayed and rotten regions of infected samples and healthy regions of non-infected samples. The classification accuracy of rotten, decayed and healthy regions at 4 and 5 days after fungal inoculation was 98.6 and 100% respectively using the mean and skewness of 500 nm, 800 nm, 900 nm and modified normalized difference vegetation index (MNDVI) spectra. CONCLUSION:Comparison of results between healthy and infected samples showed that early real-time detection of Penicillium digitatum using multispectral imaging was possible within the near-infrared (NIR) range. © 2018 Society of Chemical Industry.
Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms.
Liu Jing,Wang Shengqiang,Qin Tingting,Li Na,Niu Yuhui,Li Dandan,Yuan Yongze,Geng Hui,Xiong Li,Liu Deli
BACKGROUND:Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes. METHODS:Total RNA of P. digitatum strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. RESULTS:We present a large scale analysis about the transcriptome data of P. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR. CONCLUSIONS:The sequencing-based transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum.
Antifungal activity of β-carbolines on Penicillium digitatum and Botrytis cinerea.
Olmedo Gabriela M,Cerioni Luciana,González M Micaela,Cabrerizo Franco M,Rapisarda Viviana A,Volentini Sabrina I
β-carbolines (βCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six βCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic βCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-βCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.
Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum.
Garrigues Sandra,Gandía Mónica,Marcos Jose F
Applied microbiology and biotechnology
Antifungal proteins (AFPs) of fungal origin have been described in filamentous fungi. AFPs are small, highly stable, cationic cysteine-rich proteins (CRPs) that are usually secreted in high amounts and show potent antifungal activity against non-self fungi. The role of AFPs in the biology of the producer fungus remains unclear. AFPs have been proposed as promising lead compounds for the development of new antifungals. The analyses of available antifungal CRP sequences from fungal origin and their phylogenetic reconstruction led us to propose a new classification of AFPs in three distinct classes: A, B and C. We initiate for the first time the characterization of an AFP in a fungal pathogen, by analysing the functional role of the unique afpB gene in the citrus fruit pathogen Penicillium digitatum. Null ΔafpB mutants revealed that this gene is dispensable for vegetative growth and fruit infection. However, strains that artificially express afpB in a constitutive way (afpB (C)) showed a phenotype of restricted growth, distortion of hyphal morphology and strong reduction in virulence to citrus fruits. These characteristics support an antifungal role for AfpB. Surprisingly, we did not detect the AfpB protein in any of the P. digitatum strains and growth conditions that were analysed in this study, regardless of high gene expression. The afpB (C) phenotype is not stable and occasionally reverts to a wild type-like phenotype but molecular changes were not detected with this reversion. The reduced virulence of afpB (C) strains correlated with localized fruit necrosis and altered timing of expression of fruit defence genes.