Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Characterisation of husk rot in macadamia

The causal agent of husk rot of macadamia is often attributed to Colletotrichum gloeosporioides sensu lato. However, in recent husk rot outbreaks, the characteristic concentric ring of pycnidia of C. gloeosporioides that is associated with the disease was often absent. Due to its sporadic occurrence, the importance of husk rot is often underrated and attributed to environmental and physiological factors. In order to determine the significance, prevalence and factors that influence husk rot in macadamia, this study examined the aetiology of husk rot in Australia. The relative incidence and severity of husk rot was evaluated in several macadamia orchards over eight consecutive years. Pathogenicity assays were developed to confirm the identity of the causal agent. A range of fungi from several genera including: Diaporthe, Lasiodiplodia, Colletotrichum, Pestalotiopsis, Aspergillus, Alternaria, Nigrospora and Epicoccum were isolated from samples of macadamia pericarps with husk rot symptoms from different orchards. Fungi in the genus Diaporthe were most frequently isolated, often from symptomatic fruit. Results from pathogenicity trials showed the characteristic soft or spongy black lesions characteristic of husk rot symptoms in wounded fruits that were incubated with the diseased fruit or inoculated with a conidial suspension of Diaporthe spp. Our results suggest that injury to the macadamia fruit pericarp not only predisposes the pericarp to pathogen infection but it is a prerequisite for infection. Large variations in husk rot severity were observed over years. Husk rot severity was linked to days after anthesis and was associated with mean weekly relative humidity and minimum temperatures. This study confirmed that Diaporthe species cause husk rot in macadamia, hence, a rationale for adopting Phomopsis husk rot as the name of the disease is discussed.     

The genus <Emphasis Type="Italic">Diaporthe</Emphasis>: a rich source of diverse and bioactive metabolites

The genus Diaporthe (asexual state: Phomopsis) comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions. Although species of Diaporthe have in the past chiefly been distinguished based on host association, studies have confirmed several taxa to have wide host ranges, suggesting that they move freely between hosts, frequently co-colonizing diseased or dead tissue, while some species are known to be host-specific. They are also very frequently isolated as endophytes of seed plants. Due to their importance as plant pathogens, the genus has been thoroughly investigated for secondary metabolites, including during screening programs aimed at the discovery of novel bioactive natural products, but the respective information has never been compiled. Therefore, we have examined the relevant literature to explore and highlight the major classes of metabolites of Diaporthe and their Phomopsis conidial states. These fungi predominantly produce a large number of polyketides, but cytochalasins and other types of commonly encountered fungal secondary metabolites are also predominant in some species. Interestingly, not a single metabolite which is also known from the host plant has ever been isolated as a major component from an endophytic Diaporthe strain, despite the fact that many of the recent studies were targeting endophytic fungi of medicinal plants.     

Unravelling Diaporthe species associated with Camellia

The genus Diaporthe (syn. Phomopsis) comprises important pathogens, endophytes or saprobes with diverse host associations and worldwide distribution. Phomopsis theae is the first and hitherto the only recorded Diaporthe species on Camellia in China. The aim of this study was to investigate the Diaporthe species associated with symptomatic and asymptomatic tissues of Camellia spp. from several provinces in China. Eighty-three strains were isolated in the present study. Based on the multi-locus (ITS, HIS, TEF1, TUB) phylogenetic analyses and phenotypic characters, four novel species (D. apiculata Y.H. Gao & L. Cai, D. compacta Y.H. Gao & L. Cai, D. oraccinii Y.H. Gao & L. Cai, D. penetriteum Y.H. Gao & L. Cai), and three known species (D. discoidispora, D. hongkongensis, D. ueckerae) were identified. Five strains were assigned to D. amygdali species complex and 17 strains to D. eres species complex respectively, but they could not be further identified to species level using current multi-locus phylogenetic analysis and morphological characters. Of the identified species, D. compacta and D. discoidispora are only known as endophytes. Diaporthe hongkongensis is the dominant species on Camellia, accounting for 53.3% of the frequency of occurrence. Diaporthe lithocarpus is synonymized with D. hongkongensis; D. miriciae is synonymized with D. ueckerae. Our study revealed a high diversity of undescribed Diaporthe species on Camellia.     

Reaction of Diaporthe longicolla to a strain of Sarocladium kiliense

Phomopsis seed decay of soybean [Glycine max (L.) Merr.] is a seedborne fungal disease caused by Diaporthe longicolla that causes yield losses and reduced seed quality. Biocontrol of this pathogen by a strain of Sarocladium kiliense isolated from a culture of D. longicolla was investigated in vitro and in vivo. A zone of inhibition formed between the two fungi in vitro, but was poorly sustained, and inhibition of conidial germination of D. longicolla by a culture filtrate of S. kiliense was not significant, though D. longicolla mycelial growth was inhibited by a 25% culture filtrate. Co-inoculation of both fungi failed to reduce seed rot or increase seed germination in greenhouse and growth chamber experiments, respectively. Co-inoculation of both fungi also failed to reduce pycnidial development in colonies of D. longicolla growing on leaf pieces, but soaking of the leaf pieces with S. kiliense conidia for one or three days prior to inoculation with D. longicolla eliminated pycnidial development completely. Although it was not possible to reproduce the parasitism of S. kiliense on D. longicolla under the experimental and environmental conditions used, the potential to use S. kiliense as a protectant biocontrol for soybean fungal pathogens should be further investigated.     

Dermatophytes isolated from domestic animals in Barcelona,Spain

A comparative study was conducted with soybean material presenting symptoms of the Diaporthe/Phomopsis complex that was collected in two distant geographical regions of the world: Beltsville, MA, USA, and Vojvodina, Yugoslavia. Contrasting with earlier findings, great variability in the disease symptoms was observed, and one or more Phomopsis species could be isolated from lesions presenting similar characteristics. Among the thirty-three isolates obtained from the lesions the following species were identified: D. phaseolorum var. caulivora, P. phaseoli (teleomorph D. phaseolorum var. sojae, rare), P. longicolla (found for the first time on the soybean fields of Yugoslavia), Phomopsis sp., and one culture showing intermediate characters of D. phaseolorum var. caulivora and D. phaseolorum var. sojae. Much diversity was also found in the cultural characters of the the isolates from both localities, presumably indicating evolutionary and adaptation processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

云南元江干热河谷五种优势植物的内生真菌多样性

从蔓草虫豆（Atylosia scarabaeoides）、余甘子（Phyllanthus emblica）和黄花稔（Sida acuta）等5种云南元江干热河谷植物的525个组织块中,共分离得到内生真菌371株,内生真菌的分离频率在0.61～0.92之间,且所有植物叶内生真菌的分离频率都明显高于茎（P<0.05）。经形态学鉴定,内生真菌分属于拟茎点霉属（Phomopsis sp.）、离蠕孢属（Bipolaris sp.）和交链孢属（Alternaria sp.）等32个分类单元。拟茎点霉属为干热河谷植物优势内生真菌属,从所有被调查植物的茎叶中都分离得到该属真菌,且相对分离频率高达12.90%～50.54%。内生真菌群落组成的多样性和相似性分析结果表明,云南元江干热河谷植物内生真菌多样性偏低、宿主专一性较小。     

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.     

Acrocalymma medicaginis and Phomopsis sp. as Causal Agents of Crown Rot of Lucerne in Australia

Fungi associated with crown rotting were isolated from 3 year-old lucerne plants growing in sandy and loam soils in an irrigated stand, declining in productivity, at Langhorne Creek, South Australia. The method of isolation markedly affected the range of species recovered. Fusarium spp. and Phomopsis sp. were isolated from washed and surface sterilized crowns, whereas a larger range of fungi, especially Acrocalymma medicaginis, was isolated from unwashed crowns. A. medicaginis was most frequently isolated from rotted tissue flecked with red, while a species of Phomopsis was the dominant fungus in whitish tissue containing zone lines, especially in crown branches. A. medicaginis and Phomopsis sp. caused crown rotting of mature, wounded lucerne plants at temperatures equivalent to summer conditions at Langhorne Creek. There was no evidence that Fusarium oxysporum or F. solani, two of the most frequently isolated species, were pathogens of mature lucerne plants.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Effect of the stem blight pathogen, Phomopsis emicis, and the weevil, Perapion antiquum, on the weed Emex australis

The stem blight pathogen, Phomopsis emicis, and the weevil, Perapion antiquum, are two potential biological control agents for the annual weed, Emex australis. Neither pathogen nor weevil affected the development of rosette (5-wk-old) E. australis, but both significantly reduced stem length and number of new fruits in mature plants (10-wk-old) in growth room experiments. Stems grew two-thirds less than controls in plants attacked by weevils, had no net increase when both weevils and fungus were present, and collapsed in the presence of the fungus alone. Attack by weevils elicited a host response that slowed the development of the disease. Phomopsis emicis and Perapion antiquum reduced the ability of E. australis to produce new fruits by 77% and 68%, respectively. Pathogen and weevil together reduced the number of new fruits by 83%. Perapion antiquum did not carry or provide infection sites for P. emicis. The lack of damage by pathogen and weevil to rosettes could compromise their effectiveness as biological control agents.     

Study of die back disease incidence of neem in Karnataka,India and PCR based identification of the isolates

A disease survey of die back of neem was done in different agroclimatic regions of Karnataka, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die back were collected from different regions of Karnataka, India and they were further analysed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the detection of P. azadirachtae, the causative agent of die back of neem by polymerase chain reaction (PCR). Studies revealed the amplification of expected 141 bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Karnataka indicating the causal organism of die back disease on neem. Studies revealed a very high incidence of die back in most of the places of Karnataka. This is the first report on disease incidence of die back of neem. Hand held GPS was used in the study which helps in continuous monitoring of the diseased trees.     

A review of the phylogeny and biology of the Diaporthales

The ascomycete order Diaporthales is reviewed based on recent phylogenetic data that outline the families and integrate related asexual fungi. The order now consists of nine families, one of which is newly recognized as Schizoparmeaceae fam. nov., and two families are recircumscribed. Schizoparmeaceae fam. nov., based on the genus Schizoparme with its anamorphic state Pilidella and including the related Coniella, is distinguished by the three-layered ascomatal wall and the basal pad from which the conidiogenous cells originate. Pseudovalsaceae is recognized in a restricted sense, and Sydowiellaceae is circumscribed more broadly than originally conceived. Many species in the Diaporthales are saprobes, although some are pathogenic on woody plants such as Cryphonectria parasitica, the cause of chestnut blight, and agricultural crops such as canker diseases of soybean and sunflower caused by species of Diaporthe-Phomopsis in both temperate and tropical regions. Members of the Diaporthales such as Apiognomonia-Discula and Diaporthe-Phomopsis are commonly encountered as endophytes of woody plants.     

Synthesis and Antifungal Activity of Curcumol Derivatives

To discover novel and effective antifungal candidates, a series of new curcumol derivatives were designed, synthesized, and evaluated their antifungal activity against five phytopathogenic fungi by the mycelium growth rate method. Derivatives c4 , c22 and c23 exhibited excellent antifungal activity against Phomopsis sp. with EC₅₀ values of 3.06, 3.07, and 3.16 μM, respectively. Specifically, compound c4 exhibited the strongest antifungal activity against Phomopsis sp., which was 44 times that of pyrimethanil (EC₅₀=134.37 μM). The results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that compound c4 could cause cell senescence and death of Phomopsis sp. by changing the normal hyphal morphology and disrupting the normal metabolism of hyphal cells. Moreover, compound c4 showed excellent curative effect against Phomopsis sp. on kiwifruit. These findings confirmed that compound c4 has great potential as a potent antifungal agent.     

A survey of die-back disease of neem in Tamil Nadu,India and PCR-based confirmation of the isolates

A survey of die-back disease of neem was done in different agro climatic regions of Tamil Nadu, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die-back were collected from different regions of Tamil Nadu, India and they were further analyzed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die-back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the confirmation of P. azadirachtae – the causative agent of die-back of neem by Polymerase chain reaction (PCR). Studies revealed the amplification of expected 141bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Tamil Nadu confirming the causal organism of die-back of neem. Studies revealed a very high incidence of die-back in most of the places of Tamil Nadu. Hand held GPS was used in the study which would help in continuous monitoring of the diseased trees.     

Variation in host susceptibility among and within populations ofPlantago lanceolata L. infected by the fungusPhomopsis subordinaria (Desm.) Trav.

Summary Susceptibility toPhomopsis stalk disease ofPlantago lanceolata genotypes, sampled in three different populations with a variable degree of infection by the fungusPhomopsis subordinaria, was determined under greenhouse conditions. Susceptibility of the host varied within, but not among populations. No relationship between the intensity of the disease in the field and the mean susceptibility of the host genotypes sampled at those locations could be established. Host susceptibility appeared to be composed of the host genotypes sampled at those locations could be established. Host susceptibility appeared to be composed of different (uncorrelated) plant characteristics. Determining whether host genotypes are highly or slightly susceptible can only be achieved by field trials, where the plants are exposed to the whole set of disease inducing factors. The relevance of host susceptibility to the intensity of disease in the field is discussed in relation to the variation in pathogenicity of the fungus and the variation in environmental factors prevailing inP. lanceolata populations underP. subordinaria pathogen pressure. Grassland Species Research Group Number 123     

MoNSTR‐seq,a restriction site‐associated DNA sequencing technique to characterize Agrobacterium‐mediated transfer‐DNA insertions in Phomopsis longicolla

Phomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high‐throughput adaptation of RAD‐seq termed MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions) was developed to determine the genomic location of T‐DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens‐mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T‐DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR‐seq successfully identified a T‐DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T‐DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR‐seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.

Significance and Impact of the Study

This study describes MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions), an adaptation of restriction site‐associated DNA sequencing (RAD‐seq) to identify the position of transfer‐DNA (T‐DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high‐throughput characterization of mutants generated via Agrobacterium tumefaciens‐mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T‐DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.     

Differential Growth Inhibition of Diaporthe and Phomopsis Isolates by the Metabolic Activity of Five Actinomycetes

Fifty-five cultures derived from Diaporthe perithecia and Phomopsis pycnidia found on diverse host plant species collected at different times and sites in Vojvodina, Yugoslavia, showed distinguishing quantitative reactions to the fungistatic activity of five actinomycetes obtained as fortuitous laboratory contaminants coming from field material. Streptomyces albidoflavus , S. albus , S. diastaticus , Streptomyces sp., and Streptoverticillium sp. could be ranked by their growth-inhibitory potential, with S. albus showing the strongest, and Streptomyces sp. the lowest. The responses of the fungi varied depending on the tested actinomycetes, but two major groups could be distinguished: A, which encompased the isolates that were less affected by the proximity of the actinomycetes; and B, with those which exhibited high sensitivity in all the experiments. Group A was typically represented by Diaporthe arctii , Phomopsis longicolla, and the Phomopsis type-1 cultures from Xanthium italicum; group B was typically represented by Diaporthe/Phomopsis helianthi, Phomopsis type-2 cultures from X. italicum , and isolates from Lactuca serriola . The results obtained underscore the dissimilarities between D. arctii and D. helianthi , and corroborate the value of the physiological aspects of congeneric isolates in considering taxonomic problems in the coelomicete genus Phomopsis.