Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

Isolation and identification of pathogenic fungi causing postharvest fruit rot of kiwifruit (Actinidia chinensis) in China

Kiwifruit is a very important commercial crop in China, which is the largest producer of the fruit in the world. The rapid expansion of areas of kiwifruit cultivation has resulted in the spread of postharvest rot diseases. To clarify the pathogens causing kiwifruit postharvest rots in China, 76 pure strains were isolated from 138 rotten fruits during the shelf‐life period, with fruit collected from the 11 main regions of kiwifruit cultivation (Shaanxi, Sichuan, Henan, Guizhou, Hubei, Anhui, Jiangxi, Hunan, Fujian, Zhejiang and Jiangsu provinces) during 2014–2015. By examining the morphological and microscopic characteristics together with the results of pathogenicity testing and ITS (internal transcribed spacer) sequencing, four species were identified as the main pathogens causing kiwifruit postharvest rots in China. They were Phomopsis sp., Botryosphaeria dothidea, Alternaria alternata and Pestalotiopsis microspora, with identification rates of 52.6%, 23.7%, 13.2% and 10.5%, respectively. All isolates inoculated on wounded fruit were pathogenic but non‐pathogenic when peels were unwounded except B. dothidea. These findings have important implications for resistance breeding and control of kiwifruit postharvest rots in China.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Botryosphaeria spp. isolated from apple and several deciduous fruit trees are divided into three groups based on the production of warts on twigs, size of conidia, and nucleotide sequences of nuclear ribosomal DNA ITS regions

We examined the phytopathological and biological characters ofBotryosphaeria spp. isolated from apples and other deciduous fruit trees, and determined the nucleotide sequences of their rDNA ITS regions. TheBotryosphaeria isolates from deciduous fruit trees can be divided into three groups based on their production of warts on twigs, size of the conidia, and nucleotide sequences of rDNA ITS 1, ITS 2 and 5.8S rDNA. Isolates ofBotryosphaeria in ITS group A produced conidia of intermediate size and showed warts on infected twigs prior to the development of ring rot on fruit. This group was common on deciduous fruit trees in Japan as a causal agent of ring rot and wart bark diseases of apples and pears; and it appears similar to theB. dothidea from the US that was isolated from apple exhibiting white rot. The ITS group BBotryosphaeria produced small conidia and induced shoot blight without wart development prior to the development of ring rot on fruit. This group was localized on pear, persimmon, and kiwi fruit in restricted areas of Japan. The ITS group CBotryosphaeria consisted ofB. obtusa, the causal agent of apple black rot in the US, which produced large dark brown conidia.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture,Cell Wall Composition and Germination Kinetics

Infection with Aspergillus terreus is more likely to result in invasive, disseminated disease when compared to other Aspergillus species; importantly this species appears to be less susceptible to the antifungal drug amphotericin B. Unique to this species is the ability to produce specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this organism, we analyzed the phenotype, structure and metabolic potential of these conidia. Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC architecture by electron microscopy revealed distinct morphological differences between the two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were metabolically more active compared to PC. Additionally, AC contained less cell membrane ergosterol, which correlated with decreased susceptibility to AMB as determined using a flow cytometry based analysis. Furthermore, AC exhibited surface patches of β1-3 glucan, suggestive of attachment scarring. Collectively, the findings of this study suggest a possible role for AC in A. terreus pathogenesis.     

An integrated approach with Trichoderma harzianum DGA01 and hot water treatment on control of crown rot disease and retention of overall quality in banana

A biological control of crown rot disease of banana fruit was analysed using an integrated approach combining hot water treatment and Trichoderma harzianum strain DGA01. Treated fruits were stored at 22–25 °C and 90–95% relative humidity for 2 weeks. The bioefficacy of fungal antagonist in vitro towards crown rot-causing pathogens, namely Lasiodiplodia theobromae, Thielaviopsis paradoxa, Colletotrichum musae and Fusarium verticillioides, was enhanced by 11.41% following hot water treatment (50 °C, 20 min). DGA01 germinated on the fruit 48 h after inoculation and parasitised the pathogen. Postharvest application showed that hot water treatment and conidial suspension of DGA01 (10⁶ ml^?1) applied singly performed significantly better than the untreated control in reducing the incidence of crown rot, but were not as effective as the fungicide. The combination of hot water treatment and DGA01 gave 93% control of fruit decay which was comparable with fungicide treatment of 95%. The quality of fruit was markedly improved in hot water treatment + DGA01 as compared to those dipped in fungicide solution. The inconsistencies of single treatments, by DGA01 or hot water dips, in controlling crown rot such as variation in severity of disease among treatments and within a treatment, were lessened by dipping the fruit in DGA01 conidial suspension following hot water treatment.     

Morphology and phylogeny of <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis> causing fruit rot of olives

The taxonomic position of the causal agent of fruit rot of olives was determined from fresh collections of the fungus from central Greece. In culture it formed two types of conidia, namely fusiform, hyaline, aseptate conidia typical of the genus Fusicoccum, and dark-walled, ovoid, ellipsoid or fusiform, 1–2 septate conidia that are not typically observed in Fusicoccum. A phylogenetic analysis based on ITS and EF1- sequences placed the fungus within the same clade as Fusicoccum aesculi, which is the anamorph of Botryosphaeria dothidea, and the type of the genus Fusicoccum.     

Competition is the mechanism of biocontrol of brown rot in stone fruit by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

The mechanism of biocontrol of brown rot in stone fruit by Penicillium frequentans Westling (Pf909) was investigated using in vitro and in vivo growth assays and a benomyl-resistant strain of Monilinia fructicola (G Winter) Honey (Mf3C). For the in vitro assays, Pf909 and Mf3C conidia were suspended in Czapek-Dox broth, which was amended or not amended with a skin extract of mature peaches. The growth and germination of Pf909 and Mf3C conidia were determined by counting the number of colony-forming units on potato dextrose agar plates, which were amended or not amended with 0.5 g ml^?1 benomyl. In some of the assays, germinated Pf909 conidia were used before their exposure to Mf3C conidia. For the in vivo assays, healthy cherries were inoculated with Mf3C conidia before and after applying Pf909 conidia on the cherry surface and the incidence of brown rot was recorded for seven days. Since we found that Pf909 conidia compete with Mf3C conidia for space and nutrients in the different assays, we concluded that competition is the probable primary mechanism of biocontrol of Pf909.     

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

Fungal associations in Asphondylia (Diptera: Cecidomyiidae) galls from Australia and South Africa: implications for biological control of invasive acacias

Gall-forming Asphondylia are well represented on Australian Acacia and have potential for biological control where Australian acacias cause ecological or economic harm, particularly South Africa. Asphondylia in Australia and South Africa are associated with communities of fungi in their galls. In Australia, Botryosphaeria dothidea (as its Dichomera synanamorph) is the most abundant and sometimes the only fungus present and is implicated as the primary species forming a mutualistic relationship with Asphondylia. In the combined analysis of ITS and elongation factor 1-α sequence data, isolates of B. dothidea from Australia and South Africa form distinct sub-clades. Female Asphondylia carry B. dothidea (as Dichomera conidia) in mycangia located posterior to sternite 7. While conidia are always present on field-collected specimens, laboratory-reared females rarely carry conidia. The mechanism and location of spore collection remains unresolved, but needs to be understood if Asphondylia species are to be utilised for biological control of invasive Australian acacias. As B. dothidea is a polyphagous plant pathogen capable of infecting crops of economic importance, including Acacia plantations, the introduction of novel strains of B. dothidea associated with biological control of acacia is undesirable, however endemic forms of the fungus could possibly be exploited by introduced Asphondylia.     

Dissemination of Metarhizium anisopliae infection among the population of Odontotermes obesus (Isoptera: Termitidae) by augmenting the fungal conidia with attractants

Long-term biocontrol strategies for termite management have limited success as the behavioral response exhibited by termites in the presence of entomopathogenic fungi was different. To minimize these responses, a study was conducted to attract the termites towards the treated area by augmenting fungal conidia with a mixture of attractants such as sugarcane bagasse, sawdust and cardboard powder. In laboratory experiments, mortality due to the horizontal transmission of Metarhizium anisopliae infection among Odontotermes obesus workers and soldiers was 50 to 98% and 16 to 78% for the five isolates tested. The foraging activity of workers and soldiers increased from 23 to 58% for IWST-Ma13 when conidia were mixed with attractants. In a field study, the weight loss of stakes treated with conidial attractant decreased to 10.9 g compared to dry conidial treatments (19.3 g) for IWST-Ma13 after five months. Similarly, the width of mud galleries covering the tree bark was reduced when treated with conidial baits and gunny bags containing conidial attractant. In treated mounds, as the Acoustic emission signal voltage decreased from the threshold voltage level, the relative magnitude signal (RMS) remains low after five months of treatments with that of the control. From this study it was observed that the mixing of conidia with attractants could augment the dissemination potential of fungal infection among the termites. By adapting attractant based Metarhizium baits and dusting of conidial attractant mixture inside mounds may augment the dissemination potential in epizootic transmission of fungal infection.     

Hydrophobins from aerial conidia of Beauveria bassiana interfere with Ceratitis capitata oviposition behavior

A reduction in Ceratitis capitata Wiedemann oviposition rate was observed on fruits treated with commercial formulations containing conidia of Beauveria bassiana. In an attempt to identify the main sources of disturbance for ovipositing flies, this study investigated the possible role of different fungal fractions in relation to C. capitata oviposition behavior on treated fruits.Orange fruit treatments with different B. bassiana (Balsamo) Vuillemin preparations caused an oviposition deterrent effect in terms of reduction in the number of female visits/fruit and of oviposition punctures/fruit, in comparison to the untreated control. The most evident effects were observed for conidia-based preparations, while for the other fungal fractions assayed (mycelium and culture supernatants) these effects were moderate or negligible. These effects were concentration-dependent, and a maximum was achieved at the highest tested concentration of conidia (10⁸/ml, 10 ml per fruit).According to our results, we assumed that the physical and biochemical properties of conidia, in particular the rodlet layers of hydrophobins covering conidia surface, may impair the ability of C. capitata to detect orange-derived stimuli, such as orange odors and fruit humidity content. This hypothesis was supported by the significant decrease in the oviposition deterrent effect observed when fruits were treated with conidia deprived of the rodlet layer in comparison to equivalent suspensions of intact conidia. In addition, a suspension containing rodlet proteins alone at a concentration of 5 mg/ml also determined a significant reduction in the number of fruit visits and of oviposition punctures compared to an untreated control.     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.     

Signum, a new fungicide with interesting properties in resistance management of fungal diseases in strawberries

Signum, a new fungicide developed by BASF, was applied during 6 successive years against fungal diseases in strawberries. The product is formulated as a water dispersible granule, containing 6.7 % pyraclostrobin and 26.7 % boscalid. Pyraclostrobin is similar in chemistry to other strobilurin fungicides like kresoxim-methyl and trifloxystrobin, registered for fruit disease control. Boscalid belongs to the class of carboxyanilides. Both components in the premix formulation combine two different biochemical modes of action in the fungal cell respiration. Therefore, this co-formulation gives a broad-spectrum activity and also a reduced resistance risk for different target pathogens. Botrytis cinerea is the most important disease on strawberry-fruits and thus the control of fruit rot is mainly focused on this fungus. In average over 6 years, Signum has not only given a very good control against Botrytis fruit rot, but it has also shown a high performance in the control of Colletotrichum. Besides, Signum provides good control of powdery mildew (Podosphaera aphanis) and limits the shift to other fruit rots like leather rot (Phytophthora cactorum and leak (Rhizopus, Mucor). The availability of several categories of fungicide families with a different mode of action gives opportunities in alternating different fungicides and is the best guarantee for a sustainable control of fruit rot in all kinds of strawberry production methods. Signum should be integrated in an overall disease management program. Trials, in which the applications of Signum were timed on disease forecasting, based on environmental factors favorable for Botrytis development, were very promising. This tool can also help in establishing the IPM-concept in the production of strawberries.     

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.     

Inflammatory Monocytes Orchestrate Innate Antifungal Immunity in the Lung

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.     

Biological control of onion neck rot (Botrytis aclada): Protection of wounds made by leaf topping

The efficacy of fungal antagonists in protecting onions from neck rot caused by Botrytis aclada was investigated. Leaf wounds made by topping of onions during harvest, which are considered as important entrance sites for B. aclada, were treated with conidial suspensions (5 × 10⁷ conidia ml^‐1) of antagonists. In field experiments with artificial inoculation with conidia of B. aclada, applications of Trichoderma viride during harvest reduced the incidence of neck rot, assessed after three months’ storage of the onions at 9° C, from 35% to 24% in 1989 and from 28% to 18% in 1990, when onions initially had been stored under favourable conditions for fungal development. Comparable results were obtained with T. hamatum and Gliocladium roseum. A bioassay based on wound treatment of detached onion leaves was developed to select further antagonists. From 40 candidate antagonists tested, 20 strains gave similar or better control than the strain of T. viride applied in the field experiments. Effective antagonists could be found amongst strains of Trichoderma spp. Gliocladium spp. and Penicillium spp. as well as amongst fungi such as Aureobasidium pullulans and yeasts isolated from green leaves of onion or rye.