Antimicrobial Peptaibols,Trichokonins, Inhibit Mycelial Growth and Sporulation and Induce Cell Apoptosis in the Pathogenic Fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Trichokonins (TKs) are antimicrobial peptaibols extracted from Trichoderma pseudokoningii strain SMF2. In this paper, it was discovered that TK VI, the main active ingredient of TKs, had a profound inhibitory effect on the growth and sporulation of the moth orchid gray mold, Botrytis cinerea. In addition, TK VI increased the cell membrane permeability of the pathogen. Further investigation of nuclear DNA fragmentation, subcellular structure disintegration, and mitochondrial membrane potential depolarization, as well as the appearance of reactive oxygen species, indicated that TK VI could induce programmed cell death in the necrotrophic pathogenic fungus B. cinerea.     

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth‐promoting bacterium Azospirillum brasilense

• Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth‐promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum–strawberry plant interaction.
• Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO₃, an inhibitor of ET biosynthesis; with 1‐aminocyclopropane‐1‐carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth‐promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT‐PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes.
• Results showed that ET acts as a rapid and transient signal in the first 12 h post‐treatment. A. brasilense REC3‐inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up‐regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling.
• Ethylene production and up‐regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

First Report of Botrytis cinerea Pers. on Salicornia bigelovii Torr. in North‐West México

Grey mould has been detected on Salicornia bigelovii Torr plants in nursery and in the wild in north‐west Mexico. Sampling of the grey mould was performed in the state of Sonora, Mexico, of wild as well as cultivated S. bigelovii plants. The samples were isolated, and based on morphology, the species was identified as Botrytis cinerea Pers. Koch's postulates were fulfilled by pathogenicity tests carried out in plated petri dishes on branches from the 3‐month‐old potted S. bigelovii plants. To our knowledge, this is the first report of the isolation and identification of the fungal pathogen B. cinerea from S. bigelovii in the north‐west Mexico.     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Improved monitoring of oriental fruit moth (Lepidoptera: Tortricidae) with terpinyl acetate plus acetic acid membrane lures

Male and female moth catches of Grapholita molesta (Busck) in traps were evaluated in stone and pome fruit orchards untreated or treated with sex pheromones for mating disruption in Uruguay, Argentina, Chile, USA, and Italy from 2015 to 2017. Trials evaluated various blends loaded into either membrane cup lures or septa. Membrane lures were loaded with terpinyl acetate (TA), acetic acid (AA) and (Z)‐3‐hexenyl acetate alone or in combinations. Two septa lures were loaded with either the three‐component sex pheromone blend for G. molesta alone or in combination with codlemone (2‐PH), the sex pheromone of Cydia pomonella (L). A third septum lure included the combination sex pheromone blend plus pear ester, (E,Z)‐2,4‐ethyl decadienoate (2‐PH/PE), and a fourth septum was loaded with only β‐ocimene. Results were consistent across geographical areas showing that the addition of β‐ocimene or (Z)‐3‐hexenyl acetate did not increase moth catches. The addition of pear ester to the sex pheromone lure marginally increased moth catches. The use of TA and AA together significantly increased moth catches compared with the use of only one of the two components. Traps with the TA/AA lure outperformed the Ajar trap baited with a liquid TA plus sugar bait. The emission rate of AA was not a significant factor affecting the performance of the TA/AA lure. The addition of TA/AA significantly increased moth catches when combined with the 2‐PH lure. The TA/AA lure also allowed traps to catch both sexes. Catch of C. pomonella with the 2‐PH lure was comparable to the use of codlemone; however, moth catch was significantly reduced with the 2‐PH/PE lure. Optimization of these complex lures can likely further improve managers’ ability to monitor G. molesta and help to develop multispecies tortricid lures for use in individual traps.     

Baseline sensitivity of Botrytis cinerea to fluazinam and cross‐resistance

Grey mould, caused by the fungus Botrytis cinerea Pers ex Fr., is a very destructive and important disease worldwide. Fluazinam is a phenylpyridinamine fungicide with broad‐spectrum activities. The baseline sensitivity of B. cinerea to fluazinam is yet to be established in Henan Province, China. In this study, a total of 117 field isolates of B. cinerea were collected from 49 commercial greenhouses in different locations of Henan Province, in 2016, and the sensitivities of these isolates to fluazinam were determined based on mycelial growth. The effective concentration for 50% (EC₅₀) values ranged from 0.0038 to 0.0441 μg/ml, and the mean EC₅₀ value was 0.0201 ± 0.0081 μg/ml for mycelial growth. The frequency distribution range presented a unimodal curve. To define the cross‐resistance relationships, the linear correlation coefficients of the EC₅₀ values between fluazinam and carbendazim, procymidone, pyrimethanil or boscalid were analysed. The results showed that no correlation was observed between fluazinam and the other tested fungicides. These results provide important information to growers for the prevention and control of grey mould.     

Gamma irradiation‐induced disease resistance of pear (Pyrus pyrifolia “Niitaka”) against Penicillium expansum

In this study, the effects of gamma irradiation on the resistance of pear fruit against Penicillium expansum, the causal agent of blue mould disease, were investigated. A low dose of gamma irradiation for 14 days increased the disease resistance and firmness of pear fruits. Remarkably, exposure to 200 Gy of gamma irradiation significantly maintained fruit firmness, markedly reduced disease incidence and enhanced the activity of defence‐related enzymes (e.g., β‐1,3‐glucanase, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase) and expression of pathogenesis‐related (PR) genes (e.g., PR‐1, PR‐3 and PR‐4). Therefore, the gamma irradiation‐induced resistance against P. expansum involves both metabolic changes and the induction of expression of defence‐related genes. In addition, scanning electron microscopic analysis revealed that gamma irradiation significantly inhibits the growth of P. expansum. These results suggest that exposure of mature harvested pear fruits to artificial gamma irradiation confers fungal disease resistance; therefore, gamma irradiation represents an important strategy for controlling postharvest diseases in pear fruit.     

Variability in the efficacy of sex pheromone lures for monitoring oriental fruit moth (Lepidoptera: Tortricidae)

Studies were conducted in Chile and the United States to compare the attractiveness of various commercial sex pheromone lures and two experimental lures for oriental fruit moth, Grapholita molesta (Busck), in peach orchards treated with or without sex pheromone dispensers. The experimental lures contained the three‐component sex pheromone blend of G. molesta: Z‐8‐dodecenyl acetate, E‐8‐dodecenyl acetate and Z‐8‐dodecenol (Z8‐12:OH), and the sex pheromone of codling moth, Cydia pomonella (L.), (E,E)‐8,10‐dodecadien‐1‐ol, (codlemone). Commercial lures varied in their substrate, initial loading and blend ratio of components. Significant differences in male catches were found among commercial lures in orchards treated with or without sex pheromone dispensers. Experimental lures with the addition of codlemone significantly increased the catches of G. molesta using lures loaded with 0%, 1% or 5% Z8‐12:OH in the G. molesta blend compared with the same ratio of components in just the G. molesta blend. The experimental lures were significantly more attractive than all commercial lures in the untreated orchard. However, moth catch with the experimental lures in the sex pheromone‐treated orchard was only intermediate among all of the lures tested. These findings highlight the need to develop more effective and standardized lures that can be used in trap‐based monitoring programme for this important pest.     

Sooty Mould on Hibiscus rosa‐sinensis Caused by Leptoxyphium kurandae is Associated with Extrafloral Nectaries

In August 2013, sooty mould was observed on Chinese hibiscus (Hibiscus rosa‐sinensis) in a propagation nursery in Seoul, Korea. The sooty mould initially developed at the junction between the leaf blade and leaf petiole and then dispersed along the vein on the abaxial surface. The fungal growth pattern on the plants was quite different from general sooty moulds growing on honeydew secreted by insects on the plants. On the basis of the morphological characteristics and phylogenetic analysis using the internal transcribed spacer rDNA, this fungus was identified as Leptoxyphium kurandae. A pathogenicity test was carried out to fulfil Koch's postulates. Through field observation and a pathogenicity test, we found an association between the sooty mould and extrafloral nectaries. To our knowledge, this is the first report of sooty mould caused by L. kurandae on the extrafloral nectaries of H. rosa‐sinensis.     

Antagonistic Potential of Bacillus pumilus L1 Against Root‐Knot Nematode,Meloidogyne arenaria

This study was conducted to estimate the potential of Bacillus pumilus L1 against root‐knot nematode, Meloidogyne arenaria, in both in vitro and in vivo conditions. B. pumilus L1 was found to produce both protease and chitinase. When various concentrations (1–10%) of the bacterial culture (BC) or 0.02–0.11 mg/ml of the crude enzymes produced by B. pumilus L1 were used to treat M. arenaria eggs and second‐stage juveniles (J2), inhibition of hatching and J2 mortality were significantly increased under in vitro conditions. In addition, the hatching inhibition and J2 mortality rate were improved with increasing concentrations of BC and the crude enzymes. Similarly, these effects also increased over time after treatment with BC. Moreover, the crude enzymes caused partial degradation of the eggshell and juvenile body when treated at 0.11 mg/ml. The pot experiment also demonstrated that the application of BC to potted soil caused significant reduction in the number of galls and egg masses in the plant roots and of the J2 population as compared to the untreated control 6 weeks after M. arenaria infestation. In addition, the simultaneous application of BC upon nematode inoculation proved more effective than application 2 days postinoculation with nematode. B. pumilus L1 inoculation (BC, BCs and BC2) also promoted tomato plant growth as compared to the controls (TW, Ne, GM and NeT). Thus, our results demonstrated the ability of B. pumilus L1 as a potential biocontrol agent against root‐knot nematode, with additional activity as a plant growth promoter for tomato.     

Establishment and molecular characterization of decitabine‐resistant K562 cells

The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

Potassium phosphites in the protection of common bean plants against anthracnose and biochemical defence responses

The aim of this study was to investigate the effectiveness of potassium phosphites for the control of anthracnose and the mode of action of these products on common bean plants against Colletotrichum lindemuthianum, comparing it with the standard resistance inducer acibenzolar‐S‐methyl. The protection of plants against anthracnose was evaluated in greenhouse after treatment with potassium phosphites (Phosphite A and B, 5.0 ml/L), acibenzolar‐S‐methyl (0.25 g/L), or no treatment (control). Two sprayings of the treatments were performed, respectively, at V4 stage (three trifoliate leaves) and at the R5 stage (flower buds present). The inoculation with C. lindemuthianum was performed 5 days after the first spraying. Phosphite formulations A and B reduced the severity of anthracnose by 68.7% and 55.6%, respectively, and the presence of phosphites in the leaf tissues were detected at concentrations between 1 and 3 mm by 7 days after spraying. These same concentrations of phosphites reduced the mycelial growth of C. lindemuthianum in vitro by 15.0% to 25.7%. In addition, the activities of defence enzymes and the levels of phenolic compounds and lignin were assessed. Phosphite treatments enhanced the activity of various enzymes, including superoxide dismutase, peroxidase, chitinase, and β‐1,3‐glucanase, and increased the lignin and a small increase in the levels of soluble phenolics. This study provides evidence that phosphite treatments control anthracnose by acting directly on C. lindemuthianum and by inducing the production of defence responses.     

Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.