Blue Mold Research Articles

Antifungal activity of volatile organic compounds produced by Pseudomonas fluorescens ZX and potential biocontrol of blue mold decay on postharvest citrus

In this research, postharvest citrus fruits with blue mold caused by Penicillium italicum were treated with volatile organic compounds (VOCs) produced by Pseudomonas fluorescens ZX incubated on NA and in NB. Treatments with pure individual components of the VOCs were also conducted. The VOCs from P. fluorescens ZX inhibited mycelial growth and conidial germination of P. italicum by 42.14% and 77.86%, respectively. In vivo experiments revealed that blue mold disease incidence and lesion size upon fruits were significantly suppresed by VOCs from P. fluorescens ZX incubated on NA, in NB and on healthy fruits. In addition, the VOCs produced by bacterial fluid provided higher biocontrol efficacy than did that from NA plates. The exposure to VOCs from bacterial fluid of P. fluorescens ZX resulted in morphological abnormalities of the conidia and hyphae of P. italicum. In vitro testing of the individual pure compounds comprising the VOCs suggested that organic acids and sulfur compounds are the active components of VOCs, with dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) exhibiting the highest antifungal activity: concentrations of only 100 and 10 μL/L, respectively, afforded complete inhibition of blue mold on citrus fruits in vivo. Organic acids showed promise for controlling blue mold, but only in sufficiently low concentrations so as not to promote physiological disease or damage in the fruits.

Review of the Impact of Apple Fruit Ripening, Texture and Chemical Contents on Genetically Determined Susceptibility to Storage Rots.

Fungal storage rots like blue mould, grey mould, bull’s eye rot, bitter rot and brown rot destroy large amounts of the harvested apple crop around the world. Application of fungicides is nowadays severely restricted in many countries and production systems, and these problems are therefore likely to increase. Considerable variation among apple cultivars in resistance/susceptibility has been reported, suggesting that efficient defence mechanisms can be selected for and used in plant breeding. These are, however, likely to vary between pathogens, since some fungi are mainly wound-mediated while others attack through lenticels or by infecting blossoms. Since mature fruits are considerably more susceptible than immature fruits, mechanisms involving fruit-ripening processes are likely to play an important role. Significant associations have been detected between the susceptibility to rots in harvested fruit and various fruit maturation-related traits like ripening time, fruit firmness at harvest and rate of fruit softening during storage, as well as fruit biochemical contents like acidity, sugars and polyphenols. Some sources of resistance to blue mould have been described, but more research is needed on the development of spore inoculation methods that produce reproducible data and can be used for large screenings, especially for lenticel-infecting fungi.

Retracted: First report of Talaromyces pinophilus causing postharvest rot of sugar beet (Beta vulgaris) in Minnesota, USA

In August 2018, sugar beet roots with dark brown to blue lesions was observed (5% incidence) in Moorhead, Minnesota (46.8738° N, 96.7678° W). Infected sugar beet roots were collected and stored in a cold room at 8 ±2°C, with 78% relative humidity. A week later, dark greyish to blue mould was found on 10% of sugar beet root surface. In the second week, blue-green mycelial growth was observed on the surface (Fig. 1). Infected root tissues were dissected and small pieces (5 mm2) were disinfected using 70% ethanol for 1 minute, flushed thrice with sterile water, air dried, transferred to 50% potato dextrose agar and incubated at 25°C with a 12-hr photoperiod for seven days (Khan et al., 1). The fungal colony was observed with blue-green velvety and white margins on the periphery (Fig. 2). Conidia were hyaline, globose, and conidiophores densely penicillated (Fig. 3). The morphological characteristics of the fungus suggested a Talaromyces species (Yilmaz et al., 4). Five isolates were developed by the single spore isolation method and genomic DNA was extracted. For the PCR assay, the ITS4/ITS5 primers were used to amplify the ITS genomic region. PCR products were cleaned using an E.Z.N.A® Cycle Pure Kit (Omega Bio-tek, USA) and sent for Sanger sequencing by GenScript (GenScript, Piscataway, USA). A Blastn analysis of the ITS sequences of the five isolates showed 100% alignment with Talaromyces pinophilus (Penicillium pinophilum), GenBank Accession No. AB455516.1, with an E-value of 0. The amplified sequence (539 bp) was submitted to NCBI (MK757839.1). A pathogenicity assay was done by spraying a conidial suspension (1 × 106 conidia/ml) on the surface of healthy sugar beet (cv. Marathon) and kept in a cold room at 8°C and 80% relative humidity. Twelve 22-week-old roots with two replications were used. Mock-inoculated sugar beet roots were sprayed with sterile water. Three weeks post-inoculation, all inoculated sugar beet roots were observed to have similar symptoms to those described previously (Fig. 4). The mock-inoculated beets remained disease free. Microscopic analysis of the fungus isolated from inoculated beets revealed a similar morphology to that used for inoculation, thus fulfilling Koch's postulates. Recently a Penicillium-like Talaromyces sp. has been reported to cause sucrose loss in sugar beet piles (Strausbaugh & Dugan, 3; Strausbaugh, 2). In this present study, the pathogenicity of Talaromyces pinophilus has been demonstrated in sugar beet. This is the first report of T. pinophilus causing postharvest rot in sugar beet in the USA.

New insights into the evolution of host specificity of three Penicillium species and the pathogenicity of P. Italicum involving the infection of Valencia orange (Citrus sinensis)

ABSTRACT Blue and green molds, the common phenotypes of post-harvest diseases in fruits, are mainly caused by Penicillium fungal species, including P. italicum, P. digitatum, and P. expansum. We sequenced and assembled the genome of a P. italicum strain, which contains 31,034,623 bp with 361 scaffolds and 627 contigs. The mechanisms underlying the evolution of host specificity among the analyzed Penicillium species were associated with the expansion of protein families, genome restructuring, horizontal gene transfer, and positive selection pressure. A dual-transcriptome analysis following the infection of Valencia orange (Citrus sinensis) by P. italicum resulted in the annotation of 9,307 P. italicum genes and 24,591 Valencia orange genes. The pathogenicity of P. italicum may be due to the activation of effectors, including 51 small secreted cysteine-rich proteins, 110 carbohydrate-active enzymes, and 12 G protein-coupled receptors. Additionally, 211 metabolites related to the interactions between P. italicum and Valencia orange were identified by gas chromatography-time of flight mass spectrography, three of which were further confirmed by ultra-high performance liquid chromatography triple quadrupole mass spectrometry. A metabolomics analysis indicated that P. italicum pathogenicity is associated with the sphingolipid and salicylic acid signaling pathways. Moreover, a correlation analysis between the metabolite contents and gene expression levels suggested that P. italicum induces carbohydrate metabolism in Valencia orange fruits as part of its infection strategy. This study provides useful information regarding the genomic determinants that drive the evolution of host specificity in Penicillium species and clarifies the host-plant specificity during the infection of Valencia orange by P. italicum. IMPORTANCE P. italicum GL_Gan1, a local strain in Guangzhou, China, was sequenced. Comparison of the genome of P. italicum GL_Gan1 with other pathogenic Penicillium species, P. digitatum and P. expansum, revealed that the expansion of protein families, genome restructuring, HGT, and positive selection pressure were related to the host range expansion of the analyzed Penicillium species. Moreover, gene gains or losses might be associated with the speciation of these Penicillium species. In addition, the molecular basis of host-plant specificity during the infection of Valencia orange (Citrus sinensis) by P. italicum was also elucidated by transcriptomic and metabolomics analysis. The data presented herein may be useful for further elucidating the molecular basis of the evolution of host specificity of Penicillium species and for illustrating the host-plant specificity during the infection of Valencia orange by P. italicum.

Biocontrol ability and action mechanisms of Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 against Penicillium digitatum DSM2750 and Penicillium expansum DSM62841 causing postharvest diseases.

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

Control of major citrus postharvest diseases by sulfur-containing food additives

Sodium metabisulfite (SMBS), potassium metabisulfite (PMBS), aluminum sulfate (AlS) and aluminum potassium sulfate (AlPS), common sulfur-containing salts used as food additives, were evaluated for their antifungal activity against Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii, the most economically important pathogens causing postharvest diseases of citrus fruits. In vitro radial mycelial growth was measured on potato dextrose agar (PDA) Petri dishes amended with five different concentrations of the salts (10, 20, 30, 50, 100 mM) after 7 d of incubation at 25 °C. SMBS and PMBS at all concentrations, and AIS and AIPS above 20 mM, completely inhibited the growth of these fungi. The curative antifungal activity of the four salts to control citrus green (GM) and blue (BM) molds and sour rot (SR) was evaluated on ‘Valencia’ oranges artificially inoculated in rind wounds with P. digitatum, P. italicum and G. citri-aurantii, respectively. In vivo primary screenings showed no significant antifungal activity of AlS and AlPS to control the three diseases at any dose tested, but SMBS and PMBS reduced the incidence and severity of GM, BM and SR at various concentrations. Effective salts and concentrations were selected for in vivo dip treatments in small-scale trials. Dips at room temperature (20 °C) in SMBS and PMBS at 20 and 50 mM for 60 or 120 s significantly reduced the incidence and severity of GM and BM, with PMBS at 50 mM for 120 s the most effective treatment. Conversely, dips in SMBS and PMBS at 50 mM for 60 or 120 s did not reduce SR incidence and severity. SMBS and PMBS treatments are potentially new tools to be included in reduced-risk non-polluting strategies to control Penicillium diseases, but not SR, on citrus fruits.

Association between blue mold resistance and qPCR-based molecular markers in apple

Association between blue mold resistance and qPCR-based molecular markers in apple

Evaluation of different substrate supplements on growth and yield of oyster mushroom (Pleurotus florida)

Six different substrates viz., wheat straw, maize cob, paddy straw, banana pseudo stem, lentil straw and water hyacinth were used with suitable supplements to evaluate the average period of spawn run, pin head initiation, time taken for harvesting of flushes, fruit yield and biological efficiency of Pleurotus florida during crop season 2014 and 2015. During 2014, highest fruit yield (380.3 g/500 g) and biological efficiency (74.3%) were recorded when wheat straw supplemented with rice bran @ 2% of the substrate weight + corn flour @ 25 g/kg prepared straw + gypsum @ 20 g/kg straw among various substrates used. It also performed better in relation to time taken for spawn run (9.3 days), pin head initiation (11.7 days), time taken for harvesting of flushes (Ist flush-20.5, IInd flush-26.7, IIIrd flush-32.9). The second-best treatment was paddy straw supplemented with rice bran @ 2% of the substrate weight + corn flour @ 25 g/kg prepared straw + gypsum @ 20 g/kg. The same experiment was repeated during the crop year 2015. Surprisingly wheat straw supplemented with rice bran @ 2% of the substrate weight) + corn flour @ 25 g/kg prepared straw + gypsum @ 20 g/kg prepared straw was found superior again over other treatments with more or less similar performance. More or less similar pattern observed in rest of the treatments too. The incidence of mould infection in mushroom was also noticed in accordance with the kinds of substrates used during both crop season 2014 and 2015. Lowest incidence of mould i.e. green mould (3.13%), black mould (3.10%) and blue mould (2.42%) was observed on wheat straw when supplemented with rice bran @ 2% of the substrate weight + corn flour @ 25 g/kg prepared straw + gypsum @ 20 g/kg straw. Similar pattern of mould incidence was detected during crop season 2015.

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.

Preparation of alginate oligosaccharide and its effects on decay control and quality maintenance of harvested kiwifruit

Alginate oligosaccharide (AOS) is a biological carbohydrate formed from the degradation of sodium alginate. AOS used in this study was enzymatically prepared and had varying degrees of polymerization (2–8). AOS applied to harvested kiwifruit stored at 25 °C inhibited gray mold, blue mold, and black rot. AOS inhibited pectin solubilization, gene expression of pectin methylesterase and polygalacturonase, and the corresponding enzyme activity of their encoded proteins in kiwifruit. In contrast, AOS induced antioxidant gene expression and enzyme activity, including catalase and superoxide dismutase. The level of total phenols and flavonoids in kiwifruit was also elevated. AOS treatment also had a beneficial effect on fruit quality. Collectively, the results indicate that postharvest treatment with AOS inhibits postharvest decay and prolongs fruit quality by suppressing cell wall degradation and eliciting antioxidants in harvested kiwifruit. AOS has the potential to be used to preserve and extend the postharvest quality of kiwifruit.

Antifungal effect and possible mechanism of curcumin mediated photodynamic technology against Penicillium expansum

Abstract Contamination of Penicillium expansum has dramatic impacts on the development of the fruit and vegetable industry. In this study, the antifungal effect and its mechanism of curcumin mediated photodynamic technology (PDT) against P. expansum spores was investigated. Results indicated that PDT inactivated 99.7 % spores and inhibited spore germination, mycelial growth and disease severity of the blue mold rot on apple fruit. PDT induced the generation of reactive oxygen species (ROS), which caused oxidative damage of spores and further disrupted cellular structure. In addition, excessive ROS triggered the antioxidant defense systems of spores. Moreover, ultimate result of cell death induced by PDT was apoptosis for short time illumination, and necrosis for long time. These results can be an asset to exploit PDT for further application in controlling harvest diseases of the fruit and vegetable.

Transcriptome analysis of Penicillium italicum in response to the flavonoids from Sedum aizoon L.

Natural products extracted from plants are an alternative method for controlling postharvest citrus blue mold, caused by Penicillium italicum (P. italicum). In this study, RNA sequence analysis was used to investigate the underlying anti-fungal mechanism of flavonoids from Sedum aizoon L. (FSAL) on P. italicum. Significant differences in 3592 genes were observed, including 2507 up-regulated and 1085 down-regulated genes between the FSAL-treated and the control groups. Furthermore, the GO and KEGG analysis results indicated that FSAL inhibited genes related to the integral components of membrane, oxidation-reduction process, mitochondrion, ribosome, and amino acid metabolism. In the presence of FSAL, the cellular constituents, including DNA and RNA were leaked from hyphae of P. italicum. Reactive oxygen species (ROS) production in P. italicum was also determined with a significant concentration-effect under the treatment of FSAL. Thus, we speculate that the inhibitory activity of FSAL on P. italicum is mainly achieved by damaging the structure of the cell membrane and cell wall, disrupting the process of mitochondrial respiratory metabolism, protein biosynthesis, and amino acid metabolism, leading to cell death. The present study provided a global perspective on the molecular mechanism of FSAL on P. italicum through transcriptome analysis, which may help develop a novel plant-derived anti-fungal agent for the blue mold of citrus.

Pretreatment of the Antagonistic Yeast, Debaryomyces hansenii, With Mannitol and Sorbitol Improves Stress Tolerance and Biocontrol Efficacy.

The effect of exogenous mannitol and sorbitol on the viability of the antagonist yeast, Debaryomyces hansenii, when exposed to oxidative and high-temperature stress was determined. Results indicated that both the 0.1 M mannitol (MT) and 0.1 M sorbitol (ST) treatments improved the tolerance of D. hansenii to subsequent oxidative and high-temperature stress. MT or ST cells had a significantly higher level of cell survival, elevated the gene expression of catalase 1 (CAT1) and copper-zinc superoxide dismutase (SOD1), as well as the corresponding enzyme activity. Treated cells also exhibited a lower accumulation of intracellular reactive oxygen species (ROS), and a higher content of intracellular mannitol and sorbitol relative to non-treated, control yeast cells, when exposed to a subsequent oxidative (30 mM H2O2) or heat (40.5°C) stress for 30 min. Additionally, MT and ST yeast exhibited a higher growth rate in kiwifruit wounds, and a greater ability to inhibit postharvest blue mold (Penicillium expansum) and gray mold (Botrytis cinerea) infections. The present study indicates that increased antioxidant response induced by mannitol and sorbitol in D. hansenii can enhance stress tolerance and biocontrol performance.

Pinocembrin-7-Glucoside (P7G) Reduced Postharvest Blue Mold of Navel Orange by Suppressing Penicillium italicum Growth.

The current study aimed to examine the in vitro and in vivo antifungal potential of pinocembrin-7-glucoside (P7G). P7G is an antifungal flavanone glycoside isolated from Ficus hirta Vahl. fruit against Penicillium italicum, a causative pathogen of blue mold disease in citrus fruit, and this study elucidates its possible action mechanism. P7G had a prominent mycelial growth inhibitory activity against P. italicum, with an observed half maximal effective concentration, minimum inhibitory concentration and minimum fungicidal concentration of 0.08, 0.2, and 0.8 g/L, respectively. The data from the in vivo test show that P7G significantly reduced blue mold symptoms and disease development of P. italicum in artificially inoculated “Newhall” navel orange. Compared to the control, increases in the cell membrane permeability of P. italicum supernatant and decreases in the intracellular constituent (e.g., soluble protein, reducing sugar, and total lipid) contents of P. italicum mycelia were identified, supporting scanning electron microscopy and transmission electron microscopy observations. Furthermore, a marked decline in both chitin and glucanase contents of P. italicum mycelia treated with P7G was induced by increasing its related degrading enzyme activities, suggesting that the cell wall structure was destroyed. The current study indicated that P7G may be a novel alternative for reducing blue mold by suppressing mycelial growth of P. italicum via a cell membrane/wall-targeting mechanism.

Effects of INA on postharvest blue and green molds and anthracnose decay in citrus fruit

As a synthetic functional analog of salicylic acid, 2,6-dichloroisonicotinic acid (INA) is effective in inducing the host disease resistance of a plant against a pathogen. The effects of INA on controlling postharvest blue and green molds and anthracnose decay and defense-related enzymes on citrus fruits were investigated, and the ascorbic acid of naturally infected citrus flavedo was also measured. Results showed that 1.0 mmol L−1 INA treatments significantly reduced blue and green molds and anthracnose decay development on both wound-inoculated fruit and naturally-infected fruit compared with the control fruit. The treatment effectively enhanced the β-1,3-glucanase (GLU), chitinase (CHI), phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities and the polyphenol oxidase (PPO) in flavedo. The results presented here suggest that INA might be used as a chemical fungicide substitution to control postharvest diseases in citrus fruits.

P-Anisaldehyde Exerts Its Antifungal Activity Against Penicillium digitatum and Penicillium italicum by Disrupting the Cell Wall Integrity and Membrane Permeability.

Penicillium digitatum and P. italicum are the two important postharvest pathogens in citrus, causing about 90% of the total loss of citrus fruit during storage and transportation. Natural fungicides such as essential oils have been widely used instead of chemical fungicides for preventing and controlling postharvest diseases. In this research, p-anisaldehyde exhibited a strong inhibitory effect on P. digitatum and P. italicum, with the minimum inhibitory concentration and minimum fungicidal concentration values of both being 2.00 μl/ml. Additionally, p-anisaldehyde visibly inhibited both the green mold and blue mold development of citrus fruits inoculated with P. digitatum and P. italicum. The mycelia morphologies of these pathogens were greatly altered, and the membrane permeability and cell wall integrity of mycelia were severely disrupted under p-anisaldehyde treatment. These results suggest that the antifungal activity of p-anisaldehyde against P. digitatum and P. italicum can be attributed to the disruption of the cell wall integrity.

Bio-control activity of Pichia anomala supplemented with chitosan against Penicillium expansum in postharvest grapes and its possible inhibition mechanism

Blue mold decay caused by Penicillium expansum is one of the recently identified diseases of grapes (Vitis vinifera). The increasing concern about the use of chemical substances and pesticides in postharvest fruit pushes the trends of research toward bio-control strategies which are more sustainable and eco-friendly. This study determined the bio-control efficacy of Pichia anomala alone and supplemented with 1% chitosan in the grapes against blue mold disease. The result showed that chitosan (1%) better induced the bio-control efficacy of P. anomala. P. anomala supplemented with 1% w/v chitosan significantly reduced the disease incidence, lesion diameter and natural decay of grapes without affecting the fruit quality compared to the control. In addition it enhanced the activities of disease defence related enzymes like ascorbate peroxidase (APX) and chitinase (CHI); and decreased the formation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) which are responsible for fruits deterioration. The scanned electron microscope (SEM) concisely illustrates how the high denser yeast cells on the wounds reduced the growth of P. expansum mycelia. So it can be concluded that, P. anomala alone or P. anomala supplemented with 1% w/v chitosan can be presented as a potential bio-control alternative against the postharvest blue mold of grapes.

Transcriptome analysis of fungicide-responsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz

BackgroundPenicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified.ResultsThe present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses.ConclusionsThe present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.

Introgressing blue mold resistance into elite apple germplasm by rapid cycle breeding and foreground and background DNA-informed selection

Apple blue mold causes significant postharvest economic losses worldwide. A blue mold resistance locus, qM-Pe3.1, was previously identified on chromosome 3 of Malus sieversii PI 613981, a wild accession with inferior fruit quality. Introgression of the resistance allele into elite breeding germplasm is difficult and success of introgression and the effect of the PI 613981 genome on fruit quality cannot be phenotypically evaluated until fruiting, which occurs approximately 5 years from seed. In this study, introgression of the qM-Pe3.1 resistance allele was achieved by rapid cycle breeding, utilizing the transgenic line T1190 constitutively expressing the BpMADS4 early-flowering gene. This was supported by DNA-based diagnostic information that enabled marker-assisted selection for blue mold resistance using a locus-specific DNA test developed to detect the qM-Pe3.1 resistance allele in offspring (foreground selection). Of 75 second-generation ([‘Gala’ × PI 613981] × T1190) offspring carrying BpMADS4, 43 also carried the qM-Pe3.1 resistance allele. DNA tests for other trait loci were used to identify other desirable alleles related to fruit quality in progeny and 6874 genome-wide SNP markers (from an apple 20K Illumina® SNP array) were used to identify undesirable genomic segments of PI 613981 (background selection). Three individuals identified with favorable recombination close to qM-Pe3.1 and less than 25% of M. sieversii unimproved genome were selected as best suited for the elimination of unimproved DNA segments in subsequent generations. Our pipeline for introgression of qM-Pe3.1, facilitated by marker-assisted foreground and background selection, successfully advanced this promising germplasm in readiness for the next generation.

Silencing Dicer-Like Genes Reduces Virulence and sRNA Generation in Penicillium italicum, the Cause of Citrus Blue Mold.

The Dicer protein is one of the most important components of RNAi machinery because it regulates the production of small RNAs (sRNAs) in eukaryotes. Here, Dicer1-like gene (Pit-DCL1) and Dicer2-like gene (Pit-DCL2) RNAi transformants were generated via pSilent-1 in Penicillium italicum (Pit), which is the causal agent of citrus blue mold. Neither transformant showed a change in mycelial growth or sporulation ability, but the pathogenicity of the Pit-DCL2 RNAi transformant to citrus fruits was severely impaired, compared to that of the Pit-DCL1 RNAi transformant and the wild type. We further developed a citrus wound-mediated RNAi approach with a double-stranded fragment of Pit-DCL2 generated in vitro, which achieved an efficiency in reducing Pi-Dcl2 expression and virulence that was similar to that of protoplast-mediated RNAi in P. italicum, suggesting that this approach is promising in the exogenous application of dsRNA to control pathogens on the surface of citrus fruits. In addition, sRNA sequencing revealed a total of 69.88 million potential sRNAs and 12 novel microRNA-like small RNAs (milRNAs), four of which have been predicated on target innate immunity or biotic stress-related genes in Valencia orange. These data suggest that both the Pit-DCL1 and Pit-DCL2 RNAi transformants severely disrupted the biogenesis of the potential milRNAs, which was further confirmed for some milRNAs by qRT-PCR or Northern blot analysis. These data suggest the sRNAs in P. italicum that may be involved in a molecular virulence mechanism termed cross-kingdom RNAi (ck-RNAi) by trafficking sRNA from P. italicum to citrus fruits.