Phaseolus lunatus,a New Host of Xanthomonas axonopodis pv. phaseoli in Iran

Common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli (Xap) is one of the most destructive diseases limiting the production of common bean (Phaseolus vulgaris) in Iran. The disease has previously been described on common bean and mung bean from several regions of Iran, including the central plain and south‐western provinces. In this study, lima bean (Phaseolus lunatus cv. Christmas) plants are being reported as a new natural host of Xap in East Azerbaijan and West Azerbaijan Provinces, northwestern Iran. Disease symptoms consisted initially of water‐soaked spots that progressed to irregular necrotic lesions with chlorotic margins. Infection was observed to affect up to 40% of plants in the field. Identification of the pathogen was based on the biochemical and molecular characteristics, as well as the pathogenicity tests. To our knowledge, this is the first report of X. axonopodis pv. phaseoli causing common bacterial blight on lima bean plants in Iran.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

Studies on Apple Canker Disease. The Necrotic Toxins Produced by Valsa ceratosperma

Several metabolites responsible for the toxic manifestations of Valsa ceratosperma (Toda et Fries) Maire, a phytopathogenic fungus of the Japanese apple canker, have been isolated from its culture filtrate after growth on apple branch extract. Chemical and spectrometric studies revealed the products to be degradation products of phlorizin which is a dominant component distributed in leaves, stems, fruits and roots of apple. The toxic substances were identified as 3-(p-hydroxyphenyl) propionic acid, phloroglucinol, p-hydroxyacetophenone, p-hydroxybenzoic acid and protocatechuic acid. All of these compounds except p-hydroxyacetophenone were detected in the lesions of apple trees infected by V. ceratosperma. The fungus cultivated in a medium containing added phlorizin also produced the five toxic substances mentioned above. These results suggest that phlorizin is involved in the specific relationship between the host and the pathogen, indicating that the degradation products of phlorizin play important roles in the production of symptoms of infected apple trees.     

Screening of phytotoxicity of Alternaria macrospora MKP1 against Parthenium hysterophorus L.

Parthenium hysterophorus L. an exotic, pernicious weed is considered as one of the most troublesome weeds for agricultural sector by virtue of its high ecological amplitude and adaptability. Microbes and their by-products are now proved to be a worthy alternative to toxic chemicals used for weed management. Alternaria macrospora MKPI was isolated from the parthenium leaves infected with leaf blight and found pathogenic to the weed. The herbicidal potential of cell free culture filtrate of A. macrospora MKP1 has been tested against parthenium by employing detached leaf bioassay and seed germination bioassay and a significant damage was exhibited by the cultural filtrate of pathogen to the parthenium leaves and seeds.     

Cyanogenesis of Hevea brasiliensis during Infection with Microcyclus ulei1)

Eight Hevea species have been shown to be cyanogenic. They all liberated HCN following mechanical tissue injury. Infection of Hevea leaves with conidia of the plant pathogen Microcyclus ulei leads to a large reduction of hydrocyanic acid potential, while only small amounts of HCN are set free from the leaves into the atmosphere. HCN production by infected leaves follows a reproducible pattern with a maximum between 40 and 60 hours after infection. During the entire time of infection free HCN can be detected in the leaves. From leaves of susceptible clones high amounts of HCN are liberated whereas from resistant clones only very little HCN is released. In Hevea infections with M. ulei, cyanogenesis does not lead to defense of the fungal pathogen but impairment of the resistance reaction.     

Systemic Migration and Establishment of Xanthomonas campestris pv. pruni in Plum Leaves and Twigs

The systemic migration of Xanthomonas campestris pv. pruni (Xcp) through vascular bundles of leaves and twigs of plum was investigated. A rifampicin-resistant strain of Xcp was inoculated into leaves located midway from the tip of new green twigs of Golden King plum trees in a glasshouse. High numbers of the pathogen were recovered 4 and 8 weeks after inoculation from sections of uninoculated and symptomless veins, petioles, and twig tissue. Symptoms of bacterial spot developedwithin 8 weeks on main and secondary veins of uninoculated leaves located as far as 13 cm from the inoculated leaf on the same twig. Weekly isolation indicated the constant presence of Xcp in apparently unaffected shields of twig tissue obtained from a naturally infected Golden King orchard. Xcp apparently enters plum twigs through veins of infected leaves and migrates systemically through twigs to leaves.     

Ultrastructure of Necrotic Lesions in Nicotiana glutinosa Leaves Locally Infected with a Variant of Cucumber mosaic virus

CMV(Y/GM2)tr is a variant of Cucumber mosaic virus strain Y [CMV(Y)] which infects Nicotiana species, including N. glutinosa, to induce necrotic local lesions (NLLs) in inoculated leaves, although all other CMV strains including CMV(Y) systemically infect Nicotiana species. To investigate the morphological features of this unique host response in N. glutinosa leaves infected with CMV(Y/GM2)tr, the ultrastructure of cells surrounding completely collapsed NLLs in virus‐inoculated N. glutinosa leaves was compared with that of normal cells of mock‐inoculated N. glutinosa leaves. The changes, which have been reported in other several virus–host plant systems showing the hypersensitive response (HR), were frequently observed in cells surrounding the NLLs. Furthermore, clumping of the nuclear matrix within the nuclei, which is a feature of programmed cell death, also occurred in these cells. These results indicated that the HR‐like host response occurred at the fine structural level in the cells of N. glutinosa plants infected with CMV(Y/GM2)tr.     

Alternaria cassiae Alters Phenylpropanoid Metabolism in Sicklepod (Cassia obtusifolia)

Phenylpropaniod metabolism has been implicated in plant defence mechanism(s) against pathogen attack. In this study, phenylpropanoid metabolism was examined over a 72 h time course in the weed sicklepod (Cassia obtusifolia) in relation to pathogenic effects of the fungus Alternaria cassiae. When 3- to 4-week old seedlings were challenged by the pathogen, extrable phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) activity was dramatically increased above that in uninfected plants severalhours after inoculation and exposure to dew. Greatest increases of enzyme activity (3-fold, specific activity basis) occurred at ca 15–23 after treatment with fungal spores. After this peak of activity, PAL activity declined with time in infectedtissue, but remained greater than in uninfected plants through 65 h after treatment. Total methanol-soluble hydroxyphenolic compound levels (PAL products) were higher in shoots (stems and leaves) of infected plants at 48–72 h. Leaves contained a higherconcentration (per gram fresh weight) of hydroxyphenolic compounds than did stems, and infected leaves exhibited a phenolic content greater than that of uninfected leaves at ca 27–72 h. Increased soluble phenolic compound production correlated with the appearance of lesions and necrotic spots on leaves and stems. UV irradiation examination and spectrofluorometric analysis of thin layer chromatographic separations of methanolic exatracts revealed a substantial increase of several components ininfected tissue 48 h after inoculation. Results support the view that PAL activity increases correlate with increased phenolic compound production in this host/pathogen interaction.     

An elicitor-and pathogen-induced cdna from potato encodes a stress-responsive cyclophilin

Differential mRNA display was used to identify pathogen-responsive, stress-related genes in potato cell suspensions treated with salicylic acid and a cell wall-derived elicitor from Phytophthora infestans. Among the positive clones identified, one was found to be expressed at a significantly higher level in elicited cells than in control cells. DNA sequencing of this amplicon revealed high homology and identified it as a potato cyclophilin cDNA. The maximum amount of the cyclophilin mRNA was found 9 to 12 h after elicitation. Cyclophilin (CyP) mRNA synthesis was also up-regulated from 12 to 24 h in potato leaves locally infected with zoospores from Phytophthora infestans. However, untreated leaves responding systemically to the pathogen showed only a weak, delayed response at 24 h post infection. The observed accumulation of potato CyP mRNA in response to salicylic acid, P. infestans elicitor and P. infestans infection, suggest that CyPs play an important role in plant stress responses.     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

Chemodiversity and Antiproliferative Activity of the Essential Oil of Schinus molle Growing in Jordan

The leaves and unripe and fully‐grown fruits of Schinus molle were collected from three geographical regions of Jordan: Amman (the Mediterranean), Madaba (Irano‐Turanean), and Sahab (Saharo‐Arabian). The hydrodistilled volatile oils of fresh and dried leaves and fruits were analyzed by gas chromatography‐mass spectrometry (GC/MS). The actual composition of the emitted volatiles was determined using Solid Phase Micro‐Extraction (SPME). α‐ and β‐Phellandrenes were the major components in all the analyzed samples. Quantitative differences were observed in the obtained essential oils (0.62–5.25 %). Additionally, cluster analysis was performed. Biologically, the antiproliferative activity of the essential oil, ethanol, and water extracts of the fruits and leaves was screened on Caco2, HCT116, MCF7, and T47D cell lines. The essential oil and ethanol extracts exhibited a dose‐dependent inhibition of cell growth with IC₅₀ ranging between 21 and 65 μg/mL. The water extract did not exhibit any antiproliferative activity against the investigated cell lines.     

Gibbago trianthemae,phaeodictyoconidial genus,cause leaf spot disease of Trianthema portulacastrum

In the years 2011–2014, a series of surveys were conducted to record infestation and search natural fungal pathogens of Trianthema portulacastrum in various states of India. Among ten different agricultural important crops, heavy infestation and percentage of occurrence were observed. Gibbago trianthema, phaeodictyconidial genus, causing leaf spot disease was found on horse purslane at different districts of Haryana, Punjab and Uttar Pradesh from infected leaves and stems. Identification was confirmed on the basis of ITS rDNA sequence analysis by Macrogen Inc., Advancing through Genomics, Korea, and from CABI International Mycological Institute, England (IMI Number 502781, 502782). Sequence has been submitted to National Centre for Biotechnology Information (NCBI) with GenBank accession number KJ825852. By in vitro inoculation on Trianthema leaves, the pathogen showed similar symptoms as occurred in nature and proved Koch’s postulates. Various stages of conidia development were observed which proved the blastic-type ontogeny and porogenous development of conidiophore cell.     

Bacterial soft rot of tomato in plastic greenhouses in Crete

During recent years a new disease has been noticed on tomatoes grown in Polythene greenhouses in Crete. Early symptoms are yellowing of the lower leaves, and a yellow brown discoloration of the pith and stem xylem. As leaves wilt and die there is progressive yellowing towards the top of the plants. A progressive disintegration of the cortical tissues follows which results in a soft rot and a longitudinal splitting of the stem running mainly upwards. Soft rot of the fruits rarely appears. Severely infected plants may wilt and die, but other less affected plants often survive and yield normally. Very vigorous plants grown under humid conditions are more susceptible. Often more than 20% of the plants are infected. Isolations were made from stem (xylem, cortex and pith), from leaf xylem and from fruits of infected tomato plants collected throughout the island from 1979 to 1985. Bacteria of the genus Erwinia and Pseudomonas were consistently isolated. On the basis of physiological and biochemical characters of 49 representative pathogenic isolates, 22 were identified as Erwinia carotovora subsp. carotovora, 10 as Erwinia carotovora subsp. atroseptica, four as Pseudomonas viridiflava and 13 as Pseudomonas fluorescens biotype I. All disease symptoms were reproduced when artificial inoculations were made with the above isolates in the laboratory (20°C and 100% r.h.) on 3–4 week tomato plants and in a commercial greenhouse on 4–5 months tomato plants. Bacteria used for inoculations were reisolated. Results indicated that the disease symptoms as described may be caused by four different bacteria species.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

An Efficient Method for the Extraction of High-Quality Fungal Total RNA to Study the <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>–<Emphasis Type="Italic">Musa</Emphasis> spp. Interaction

Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant–fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum). Aminael Sánchez-Rodríguez and Orelvis Portal contributed equally to the article.