Detailed description of developmental growth stages of Diplocarpon rosae in Rosa: a core building block for efficient disease management

Black spot disease of roses caused by the ascomycetous fungus Diplocarpon rosae teleomorph (Marssonina rosae anamorph) is a widespread and devastating disease. Despite considerable progress in the management of black spot disease in the recent years, it is still unclear by which mechanisms this fungus colonises and invades the host system, and without a good knowledge of such infection machineries, it not possible to fully overcome the challenges of D. rosae infection. By exploring research contributions up to date, we highlight in this review the ultrastructure of D. rosae infection cycle in the host cell by emphasising on several aspects related to its in vitro and in vivo germinations, the infection mechanisms of the fungus, the different fungal structures formed in the host cells and the optimum storage conditions for D. rosae to retain its viability and pathogenicity over time. Here, attention is particularly focused on the asexual life cycle of D. rosae, with the sexual cycle being briefly discussed. In addition, a new dimension of research approaches to effectively control black spot disease of roses, that is, how to accurately use the advanced biotechnology tools to speed up the current state of the disease management, is proposed here.     

Microscopic and biochemical evidence of differentially virulent field isolates of Diplocarpon rosae causing black spot disease of roses

Black spot disease caused by Diplocarpon rosae is one of the most widespread diseases of roses that are very difficult to control due to the generative reproduction and complex genetic constitution of roses and up to now the control of black spot still requires intensive use of systemic fungicides. Here we report for the first time evidence of differentially virulent field isolates of D. rosae. Using a combination of fungal structures, disease symptoms and host cells protein expression pattern analysis we here provide direct biochemical evidence that tropical field isolates of D. rosae are more virulent and caused disease symptoms earlier than their temperate counterparts. The tropical fungal field isolates strongly induced an excessive accumulation of ROS and repressed activity of pathogenesis-related proteins such as peroxidases, chitinase and phenylalanine ammonia lyase compared to their temperate counterparts. These findings bring insights into a hidden pathogenic characteristic of tropical D. rosae field isolates compared to their temperate counterparts and open a novel dimension of parameters to be considered when controlling black spot disease of roses by fungicides in tropical versus temperate regions. Interestingly, we found that treatment of rose leaves with ROS (H₂O₂) prior to fungal inoculation promoted plant defense response regardless of the isolate virulence based on protein expression pattern analysis, suggesting that ROS (H₂O₂) can be efficiently incorporated into black spot disease management.     

Rdr3, a novel locus conferring black spot disease resistance in tetraploid rose: genetic analysis, LRR profiling, and SCAR marker development

Black spot disease of rose, incited by the fungus Diplocarpon rosae, is found worldwide and is the most important disease of garden roses. A gene-for-gene interaction in this pathosystem is evidenced by the presence of pathogenic races of D. rosae and the previous discovery of a dominant resistance allele at the Rdr1 locus in the diploid Rosa multiflora. The objective of the present study was to genetically analyze resistances to North American black spot races 3, 8, and 9 previously reported in tetraploid roses. Resistance to North American races 3 and 8 segregated 1:1 in multiple F₁ populations, indicating that both are conferred by dominant alleles at single loci and are present in simplex (Rrrr) configuration. Gene pyramiding was demonstrated by combining both resistances into single genotypes. Resistance to race 9 was partial and segregated in a quantitative fashion. Analysis of these populations with microsatellite markers previously developed for Rdr1 revealed that the gene conferring race 3 resistance resides within the same R gene cluster as Rdr1. Race 8 resistance segregated independently and is, therefore, a novel locus for black spot resistance in rose which we have named Rdr3. NBS and LRR profiling were used in a bulked segregant analysis to identify a marker 9.1 cM from Rdr3, which was converted to a SCAR marker form for marker-assisted breeding.     

Influence of phylloplane colonizing biocontrol agents on the black spot of rose caused by Diplocarpon rosae

Abstract

An attempt was made to study the biocontrol efficacy of antagonistic microorganisms in phylloplane of rose cv. Edward to manage the black spot (Diplocarpon rosae) disease. Eight antagonistic microorganisms were tested in vivo against the black spot pathogen. Among these, Trichoderma viride and Pseudomonas fluorescens pf1 reduces the mycelial growth significantly. These two biocontrol agents were evaluated for their ability to induce defense-related enzymes and chemicals in plants. Increased activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO) and total phenolics were recorded in all the biocontrol agents treated leaves. P. fluorescens Pf1 recorded early and increased synthesis of the entire defense-related enzymes and total phenol within 6 days. The application of biocontrol agents induced the defense-related enzymes involved in phenyl propanoid pathway in addition to direct antagonism, which collectively contribute for enhanced resistance against invasion of Diplocarpon rosae in rose.     

The TNL gene Rdr1 confers broad‐spectrum resistance to Diplocarpon rosae

Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field‐grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single‐dominant TIR‐NBS‐LRR (Toll/interleukin‐1 receptor‐nucleotide binding site‐leucine rich repeat) (TNL)‐type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single‐spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non‐transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose–black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow.     

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.     

Bumblebee Venom Serine Protease Increases Fungal Insecticidal Virulence by Inducing Insect Melanization

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.     

Atg26-mediated pexophagy and fungal phytopathogenicity

Colletotrichum orbiculare is a plant pathogenic fungus that causes disease on cucumber plants. A homologue of ATG26 (CoATG26) was identified as the gene involved in pathogenesis. The peroxisomes are degraded via pexophagy during formation of an infection structure called the appressorium of C. orbiculare. The Coatg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step. Importantly, the autophagic degradation of peroxisomes was significantly delayed in the appressoria of the Coatg26 mutant. Domain and localization analysis of CoAtg26 also demonstrated a strong correlation of functional pexophagy with pathogenicity. Furthermore, in contrast to the Coatg26 mutant, the Coatg8 mutant, defective in the entire autophagic pathway, could not form normal appressoria in the earlier steps of morphogenesis. These results indicate that CoAtg26-mediated pexophagy plays critical roles in host plant invasion.     

Characterization of Colletotrichum orchidophilum,the agent of black spot disease of vanilla

Black spot symptoms were reported on vanilla plants in Reunion Island in 2011. They have repeatedly reduced annual pod production by 10% to 30%. The disease is characterized by dark spots that appear in slight depressions on flowers, pods, leaves and stems. The spots then develop into broad clearly depressed necrotic plaques. Morphological and molecular analyses, as well as pathogenicity tests, identified the fungus Colletotrichum orchidophilum (Ascomycota) as the causal agent of the disease. Inoculation tests in controlled conditions confirmed that the two C. orchidophilum strains isolated from fruit lesions are pathogenic on the leaves and fruits of Vanilla planifolia (accessions CR0001 and CR0020). However, these strains induced symptoms only when the epidermis of leaves and fruits had been punctured by a needle. In the absence of injury, no symptom appeared. Colletotrichum arxii and Fusarium proliferatum (Ascomycota) are fungal species that are also frequently isolated from black spot lesions. However, they are not pathogenic to vanilla. This is the first report of C. orchidophilum in Reunion Island. It is also the first demonstration of C. orchidophilum's pathogenicity to an orchid. Simple preventive control measures were proposed to reduce the incidence of black spot disease in vanilla plots.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

MoGrr1, a novel F-box protein,is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae.     

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific,toxin-producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant–pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.     

Cytoplasmic- and extracellular-proteome analysis of <Emphasis Type="Italic">Diplodia seriata</Emphasis>: a phytopathogenic fungus involved in grapevine decline

Background  

The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome.     

Host/parasite interaction in blackspot disease of roses caused by Diplocarpon rosae Wolf

The utilization of amino acids by Diplocarpon rosae Wolf was investigated and compared with amino acid compositions of leaves of resistant and of tolerant roses. No amino acid appeared to determine resistance of roses to blackspot on a nutritional basis, but phenylalanine marginally increased resistance when taken up by wounded leaves of resistant or tolerant varieties. There is an accumulation of phenolic compounds in response to infection by D. rosae. Most phenolic compounds are toxic to D. rosae in vitro, particularly ellagic acid and p-coumaric acid, but their presence and quantity in healthy rose leaves could not be related to disease resistance. Inhibition of germination on rose leaves of conidia of the fungus was determined in a manner that suggested production of a phytoalexin. It is suggested that resistance of roses to D. rosae may be related to accumulation of polyphenols and possibly to the production of a phytoalexin.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

No host‐associated differentiation in the gall wasp Diplolepis rosae (Hymenoptera: Cynipidae) on three dog rose species

Differences in quality and quantity of secondary compounds, as well as in leaf traits of host plants, may influence the host choice of herbivores. Different host preferences could lead to host‐associated differentiation, the first step of sympatric speciation. In the present study, we investigated whether the rose gall wasp Diplolepis rosae L. (Hymenoptera: Cynipidae) shows genetic differentiation related to its host plants (Rosa canina L., Rosa corymbifera Borkh., and Rosa rubiginosa L.). These three host species radiated recently and subsequently expanded their range. Therefore, we expected a diversification within the closely‐associated phytophagous insects. The process of genetic differentiation should be intensified in D. rosae by its close relationship to the host plant, as well as by its parthenogenetic reproduction (infection rate by Wolbachia sp. of almost 100%). However, using 106 polymorphic amplified fragment length polymorphism markers, we found no genetic differentiation among the wasps from different host plants. The population structuring between geographical localities was also low, suggesting considerable gene flow between sites. In part, the low genetic differentiation between sites is explained by the wide distribution of host species and hybrids between host plants. Hybrids with intermediate traits may facilitate the gene flow between wasp populations exploiting different host species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 369–377.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.     

First report of leaf spot disease caused by Corynespora torulosa MCC-1368 on cotton plant from India

In present study, the leaf spot disease of cotton plant emerged in the North Maharashtra region of India was reported. The fungal phytopathogen associated with inducing the leaf spot disease symptoms was isolated and characterised. The isolated fungus was identified as Corynespora torulosa (Deposition accession number, MCC-1368; Genbank accession no. MF462072) based on morphological and cultural characteristics and molecular analysis of ITS region. The pathogenicity of fungal phytopathogen was verified by Koch’s postulates. To our knowledge, this is the first report of incidence of leaf spot disease caused by Corynespora torulosa on cotton plant.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.