Phoma Macrostoma Research Articles

Reappraisal of Didymella macrostoma causing white tip disease of Canada thistle as a new species, Didymella baileyae, sp. nov., and bioactivity of its major metabolites

ABSTRACT Bioherbicides are expected to be a supplement to integrated pest management, assisting in the control of problematic weed species. For instance, bioherbicides (Phoma and BioPhoma) were recently registered in Canada and the USA for the control of some perennial dicotyledonous weeds in lawns. These products are based on strains of the fungus Didymella macrostoma (syn. Phoma macrostoma) that causes white tip disease (WTD) in Canada thistle (Cirsium arvense). In this study, WTD was reported for the first time in the Russian Federation. Analysis of the internal transcribed spacer (ITS) region of nuc rDNA and secondary metabolite profiling confirmed the identity of Russian WTD isolates to Canadian biocontrol strains identified as D. macrostoma. Multilocus phylogenetic analysis based on sequencing of the ITS region, partial large subunit nuc rDNA region (28S), RNA polymerase II second largest subunit gene (rpb2), and partial β-tubulin gene (tub2) has differentiated the WTD isolates from C. arvense and D. macrostoma isolates from other plant hosts. Based on phylogenetic, morphological, and chemotaxonomic features, these WTD isolates were described as a new species named Didymella baileyae, sp. nov. This study also demonstrated the low pathogenicity of the ex-type D. baileyae isolate VIZR 1.53 to C. arvense seedlings and its asymptomatic development in the leaves of aboveground shoots. The organic extracts from mycelium and culture filtrate of D. baileyae, as well as macrocidin A and macrocidin Z, displayed phytotoxicity both to C. arvense leaves and seedlings. Macrocidin A was only detected in the naturally infected leaf tissues of C. arvense showing WTD symptoms. Macrocidins A and Z demonstrated low antimicrobial and cytotoxic activities, exhibiting no entomotoxic properties. The data obtained within this study on the pathogenicity and metabolites of D. baileyae may be important for the rational evaluation of its prospects as a biocontrol agent.

First report of Didymella macrostoma causing calyx-end rot of pear (Pyrus communis) in The Netherlands.

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 μm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 μm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.

Phoma spp. an untapped treasure of cytotoxic compounds: current status and perspectives.

The genus Phoma has been explored for a wide range of secondary metabolites signifying a huge range of bioactivities. Phoma sensu lato is a major group that secretes several secondary metabolites. The genus Phoma mainly includes Phoma macrostoma, P. multirostrata, P. exigua, P. herbarum, P. betae, P. bellidis, P. medicaginis, P. tropica, and many more species from the genus that are continuously being identified for their potential secondary metabolites. The metabolite spectrum includes bioactive compounds like phomenon, phomin, phomodione, cytochalasins, cercosporamide, phomazines, and phomapyrone reported from various Phoma spp. These secondary metabolites show a broad range of activities including antimicrobial, antiviral, antinematode, and anticancer. The present review is aimed to emphasize the importance of Phoma sensu lato fungi, as a natural source of biologically active secondary metabolites, and their cytotoxic activities. So far, cytotoxic activities of Phoma spp. have not been reviewed; hence, this review will be novel and useful for the readers to develop Phoma-derived anticancer agents. KEY POINTS: • Different Phoma spp. contain a wide variety of bioactive metabolites. • These Phoma spp. also secrete cytotoxic and antitumor compounds. • The secondary metabolites can be used for the development of anticancer agents.

Curvularin Isolated From Phoma macrostoma Is an Antagonist of RhlR Quorum Sensing in Pseudomonas aeruginosa.

Quorum sensing (QS) is an attractive target for the treatment of multidrug-resistant Pseudomonas aeruginosa, against which new antibiotics are urgently needed. Because LasR is at the top of the QS hierarchy controlling Rhl and PQS systems, most QS inhibitors have been targeted to LasR. However, it has recently been reported that in clinical isolates of P. aeruginosa, LasR is frequently mutated and nonfunctional, and RhlR independently acts to produce virulent factors that maintain toxicity. Thus, for effective treatment of chronic cystic fibrosis infections, RhlR antagonists is needed to prevent the LasR-independent Rhl system, but RhlR antagonists have rarely been reported. In this study, we found that curvularin, an aromatic compound with a cyclized alkyl side chain isolated from Phoma macrostoma, at a low micromolar concentration of 1–30 μM potently and selectively inhibited pyocyanin and rhamnolipid production without affecting the cell viability of P. aeruginosa. Only high concentration (more over 100 μM) curvularin negligibly inhibited biofilm formation and elastase production, suggesting that curvularin at low concentrations selectively inhibits RhlR. The QS antagonism by curvularin was investigated in experiments using QS competition and signaling molecules assays with QS gene expression analysis, and the results showed that, indeed, at low concentrations, curvularin selectively antagonized RhlR; in contrast, it negligibly antagonized LasR only when applied at a high concentration. The exclusive RhlR antagonizing activity of curvularin at low concentrations was confirmed using QS mutants; specifically, curvularin at low concentrations inhibited pyocyanin and rhamnolipid production by selectively antagonizing N-butanoyl homoserine lactone (BHL)-activated RhlR. Moreover, by targeting RhlR, curvularin reduced the in vivo virulence of wild-type P. aeruginosa as well as lasR mutants in Caenorhabditis elegans. Overall, low-concentration curvularin is a pure RhlR antagonist in P. aeruginosa, and to the best of our knowledge, this is the first report describing an RhlR antagonist from natural resources. Hence, curvularin has great potential for the development of chronic P. aeruginosa infection therapeutics and for the study of RhlR function in the complex QS system.

Synthesis and Bioactivity of a Macrocidin B Stereoisomer.

A stereoisomer of macrocidin B, a presumed metabolite of the fungus Phoma macrostoma, was synthesized in 18 steps and 2.7% yield from protected l-tyrosine that was N-β-ketoacylated with a fully functionalized octanoyl Meldrum's acid. Dieckmann condensation gave a 3-acyltetramic acid, which was macrocyclized via Williamson etherification between the phenol and epi-bromohydrin termini. This macrocidin B stereoisomer showed a weaker herbicidal effect than macrocidin A and no similar inhibitory effect on biofilms of Staphylococcus aureus.

Macrooxazoles A-D, New 2,5-Disubstituted Oxazole-4-Carboxylic Acid Derivatives from the Plant Pathogenic Fungus Phoma macrostoma.

In our ongoing search for new bioactive fungal metabolites, four previously undescribed oxazole carboxylic acid derivatives (1–4) for which we proposed the trivial names macrooxazoles A–D together with two known tetramic acids (5–6) were isolated from the plant pathogenic fungus Phoma macrostoma. Their structures were elucidated based on high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. The hitherto unclear structure of macrocidin Z (6) was also confirmed by its first total synthesis. The isolated compounds were evaluated for their antimicrobial activities against a panel of bacteria and fungi. Cytotoxic and anti-biofilm activities of the isolates are also reported herein. The new compound 3 exhibited weak-to-moderate antimicrobial activity as well as the known macrocidins 5 and 6. Only the mixture of compounds 2 and 4 (ratio 1:2) showed weak cytotoxic activity against the tested cancer cell lines with an IC50 of 23 µg/mL. Moreover, the new compounds 2 and 3, as well as the known compounds 5 and 6, interfered with the biofilm formation of Staphylococcus aureus, inhibiting 65%, 75%, 79%, and 76% of biofilm at 250 µg/mL, respectively. Compounds 5 and 6 also exhibited moderate activity against S. aureus preformed biofilm with the highest inhibition percentage of 75% and 73% at 250 µg/mL, respectively.

Phoma macrostoma сирек кездесетін онихомикоз қоздырғышының сипаттамасы (син. Didymella macrostoma)

The article presents the results of studying the biological properties of the Phoma macrostomastrain, first described in Kazakhstan as the causative agent of onychomycosis. The authors obtained a pure culture of Phoma macrostoma coelomic, isolated from the affected nail plate of a patient in a narcological dispensary, and studied its biological properties: cultural, morphological, biochemical. Surface cultivation on differential media, microscopy in agar blocks and adhesive tape preparations, molecular genetic identification was carried out. The features of the formation of fungus colony are described, the characteristic morphological structures of the coelomycete, moderate fermentation of carbohydrates and the absence of urease activity are revealed. On the dextrose agar of Sabouraud nutrient medium, by 10-18 days, flat colonies of pale pink colour, with a pinkish-orange tint in the centre, yellowish along the edge, with a raised centre and relief radial stripes, were formed, the reverser was yellow-brown. The appearance of single growth tubes was noted within the first 12 hours, the massive division of single cells and the growth of filamentous filaments - 18 hours after sowing the spores. By the end of the second day, there is a powerful growth and active branching of the mycelium, the appearance of rudiments of pycnidia in the form of seals along the course of mature mycelial filaments, by the end of the third day, the formation of pycnidia and chlamydospores of the brown colour of various shades. Ripe transparent, unicellular, elliptical pycnospores emerge from pycnidia and located in the form of a mucous drop. The strain is characterized by moderate saccharolytic activity in relation to glucose, sucrose, mannose, mannitol, lactose, the almost complete absence of urease activity. Molecular genetic identification by the Sanger method made it possible to identify the Phoma macrostoma strain with 100% certainty and to deposit the nucleotide sequence in GenBank as isolate No. MN701978.1 Didymella macrostoma 104-Kz. Key words: biology of the pathogen, genetic identification, onychomycosis, pycnidia, coelomycetes

Характеристика редкого возбудителя онихомикоза Phoma macrostoma (син. Didymella macrostoma)

The article presents the results of studying the biological properties of the Phoma macrostomastrain, first described in Kazakhstan as the causative agent of onychomycosis. The authors obtained a pure culture of Phoma macrostoma coelomic, isolated from the affected nail plate of a patient in a narcological dispensary, and studied its biological properties: cultural, morphological, biochemical. Surface cultivation on differential media, microscopy in agar blocks and adhesive tape preparations, molecular genetic identification was carried out. The features of the formation of  fungus colony are described, the characteristic morphological structures of the coelomycete, moderate fermentation of carbohydrates and the absence of urease activity are revealed. On the dextrose agar of Sabouraud nutrient medium, by 10-18 days, flat colonies of pale pink colour, with a pinkish-orange tint in the centre, yellowish along the edge, with a raised centre and relief radial stripes, were formed, the reverser was yellow-brown. The appearance of single growth tubes was noted within the first 12 hours, the massive division of single cells and the growth of filamentous filaments - 18 hours after sowing the spores. By the end of the second day, there is a powerful growth and active branching of the mycelium, the appearance of rudiments of pycnidia in the form of seals along the course of mature mycelial filaments, by the end of the third day, the formation of pycnidia and chlamydospores of the brown colour of various shades. Ripe transparent, unicellular, elliptical pycnospores emerge from pycnidia and  located in the form of a mucous drop. The strain is characterized by moderate saccharolytic activity in relation to glucose, sucrose, mannose, mannitol, lactose, the almost complete absence of urease activity. Molecular genetic identification by the Sanger method made it possible to identify the Phoma macrostoma strain with 100% certainty and to deposit the nucleotide sequence in GenBank as isolate No. MN701978.1 Didymella macrostoma 104-Kz. Key words: biology of the pathogen, genetic identification, onychomycosis, pycnidia, coelomycetes

Occurrence and diversity of cultivable autochthonous microscopic fungi in substrates of old environmental loads from mining activities in Slovakia

Distribution and biodiversity of soil microscopic fungi in 5 areas of old environmental loads in Slovakia were studied in relation to very low amount of organic matter (%TOC from 0.2 to 3.54) and to the pH gradient from ultra-acidic (< 3.5) to very strongly alkaline (> 9.0). All soil samples were affected by several hundred years of mining activities and contained heavy metals and other toxic elements: arsenic, cadmium, copper, zinc, antimony, lead. Concentrations of toxicants highly exceeded their limited values. Fifty-three genera and 112 species of microscopic fungi were identified. Among them, Zygomycota occurred very rarely (8 genera and 12 species), except of samples with the highest content of TOC (2.01–3.54% - samples 2 and 6), regardless their pH. Though, on the other hand, from some similar samples (3, 5 and 9), incl. those with relatively high TOC (0.14–2.62%), the lower fungi were not recovered. Forty one genera and 95 species of Ascomycota represented the most abundant fungal phylum in all investigated samples. Among them, Penicillium chrysogenum var. chrysogenum, Aspergillus niger and Neosartorya fischeri were isolated the most often. Phytopathogenic moulds of Bionectria ochroleuca, Lewia infectoria, Phoma macrostoma and Phlebia acerina were also occurred frequently. The highest biodiversity of microfungal community was recorded in the extreme acidic environment, followed by the neutral, ultra-acidic and the very strong acidic ones. There was no similarity in microfungal spectrum found in the samples studied. Except of the ultra acidic and extreme acidic samples (1−2) as well as the ultra acidic and strong acidic ones (1−4) with the most rich mycobiota, that may indicate a certain similarity degree.

Isolation, structural modification of macrophin from endophytic fungus Phoma macrostoma and their cytotoxic potential

Four known metabolites, macrophin (1), rosellisin (2), 2-(2-hydroxy-5-6-methoxy-3-methylene-1,4-benzodioxin-2(3H)-one (3), and methoxyphenoxyacrylic acid (4) were isolated, for the first time, from an endophytic fungus, Phoma macrostoma inhabiting the inner tissue of medicinal plant Glycyrrhiza glabra Linn. Their structures were characterized by comparison of their NMR data with literature data, and X-ray diffraction data of macrophin (1) as described herein for the first time. The compounds (1–4) were evaluated for their growth-inhibitory activities against a panel of cancer cell lines. Of these substances, macrophin (1) showed prominent cytotoxic activity against the MDA-MB-231, T47D, MCF-7, and MIAPaCa-2 cancer-cell lines with IC50 values of 14.8, 8.12, 13.0, and 0.9 μM, respectively. This biological significance has prompted us to generate a series of five acylated analogs (1a–1e) of 1 to conduct structure–activity relationship (SAR) study. The result illustrates all the analogues (1a–1e) were more potent than 1 against MDMB-231 and MIAPaCa-2 cancer cell lines. The compound 1d, an n-butanoyl substituent, has been proven to be three-fold more potent than 1 with IC50 values of 0.3 and 3.7 µM against MIAPaCa-2 and MDMB-231 cell lines, respectively.

Phoma macrostoma : as a broad spectrum bioherbicide for turf grass and agricultural applications.

Abstract Phoma macrostoma Montagne 94-44B is an effective bioherbicide for broadleaved weed reduction. In 2016, Health Canada's Pest Management Regulatory Agency (PMRA), under the authority of the Pest Control Products Act and Regulations, granted full registration for the sale and use of the bioherbicide P. macrostoma Montagne 94-44B to control a broad spectrum of broadleaf weeds in established turfgrass and new seeding of grasses as well as in field grown nursery plants, trees and container-grown ornamentals. P. macrostoma 94-44B colonizes susceptible and non-susceptible plant roots, however, only in susceptible plants, such as Taraxacum officinale (dandelion), do mycelium proliferate around the vascular trachea interfering with the function of neighbouring cells while not entering them. Macrocidins, secondary metabolites secreted by P. macrostoma 94-44B mycelia, inhibit multiple steps in carotenoid precursor formation including phytoene desaturase and steps associated with β carotene and lutein carotenoid biogenesis, Fe and Mg chelation and OJIP chlorophyll fluorescence, thus uncoupling the light-harvesting complex of photosystem II from the reaction centre. The combined actions of P. macrostoma 94-44B and macrocidins elicit photobleaching of the leaves and eventual weed death. Research is underway to exploit Phoma's bioherbicidal actions for weed management in agriculture. P. macrostoma 94-44B can be used to reduce broadleaved weeds in crops such as wheat, corn, potato, forage grasses and established alfalfa (2nd growth or older). Soil residue studies reveal that neither P. macrostoma 94-44B nor macrocidins are present after 1 year suggesting that farmers could plant Phoma -sensitive crops such as legumes, flax or canola in subsequent years.

Alkaline Technosol contaminated by former mining activity and its culturable autochthonous microbiota

Technosols or technogenic substrates contaminated by potentially toxic elements as a result of iron mining causes not only contamination of the surrounding ecosystem but may also lead to changes of the extent, abundance, structure and activity of soil microbial community. Microbial biomass were significantly inhibited mainly by exceeding limits of potentially toxic metals as arsenic (in the range of 343–511 mg/kg), copper (in the range of 7980–9227 mg/kg), manganese (in the range of 2417–2670 mg/kg), alkaline and strong alkaline pH conditions and minimal contents of organic nutrients. All of the 14 bacterial isolates, belonged to 4 bacterial phyla, Actinobacteria, Firmicutes; β- and γ-Proteobacteria. Thirteen genera and 20 species of microscopic filamentous fungi were recovered. The most frequently found species belonged to genera Aspergillus (A. clavatus, A. niger, A. flavus, A. versicolor, Aspergillus sp.) with the dominating A. niger in all samples, and Penicillium (P. canescens, P. chrysogenum, P. spinulosum, Penicillium sp.). Fungal plant pathogens occurred in all surface samples. These included Bjerkandera adustata, Bionectria ochloleuca with anamorph state Clonostachys pseudochloleuca, Lewia infectoria, Phoma macrostoma and Rhizoctonia sp.

Selective Broadleaf Weed Control in Turfgrass with the BioherbicidesPhoma macrostomaand Thaxtomin A

Both regulatory and consumer forces have increased the demand for biopesticides, particularly in amenity areas such as turfgrass. Unfortunately, few natural products are available for selective weed control in turfgrass. Two bioherbicides reported to control broadleaf weeds without injuring turfgrass arePhoma macrostomaand thaxtomin A. Field and container experiments were conducted to evaluate PRE and POST efficacy ofP. macrostomaand thaxtomin A on regionally important broadleaf weeds. In container experiments, PRE applications ofP. macrostomaprovided 65 to 100% control of dandelion, marsh yellowcress, and flexuous bittercress, equivalent to that of pendimethalin. Control of yellow woodsorrel, henbit, hairy galinsoga, common chickweed, or annual bluegrass was less than with pendimethalin. In contrast, POST applications did not control any species as well as an industry-standard synthetic auxin herbicide. PRE or POST applications of thaxtomin A controlled six of the eight species tested as well as the industry-standard PRE or POST herbicides. In field tests, overall PRE broadleaf weed control withP. macrostomaand thaxtomin A peaked 4 wk after treatment at 64 and 72%, respectively, and declined afterward, suggesting that these bioherbicides possess short residuals and therefore must be reapplied for season-long control. Overall POST broadleaf weed control usingP. macrostomaand thaxtomin A was only 41 and 25%, respectively. PRE followed by early-POST applications of thaxtomin A provided ≥ 86% henbit control. These results suggest that bothP. macrostomaand thaxtomin A are capable of controlling certain broadleaf weeds in turfgrass. However, both lack efficacy on some important weed species, particularly chickweed. Thaxtomin A efficacy on henbit was improved by increased dose and by PRE followed by early-POST applications.

The dominant modes of action of macrocidins, bioherbicidal metabolites of Phoma macrostoma, differ between susceptible plant species

The bioherbicidal fungus Phoma macrostoma produces macrocidins, which induce chlorosis in susceptible plant species by interfering with the carotenoid biosynthesis. Macrocidins inhibit the enzyme phytoene desaturase (PDS) and likely act on other components of the carotenoid biosynthetic pathway. It was hypothesized that macrocidins’ mode(s) of action differ between susceptible plant species that respond differently to these phytotoxins. This idea was tested by exploring the impact of macrocidins on the carotenoid and carotenoid precursor profiles over time, as well as chlorophyll fluorescence parameters, symptom severity and biomass, in dandelions, groundsel and chickpea. While PDS was inhibited in all three plants, this impact was strongest in dandelion and weakest in groundsel. However, groundsel showed the most severe macrocidin-induced symptoms and biomass reduction. In solution, macrocidin A bound iron and magnesium cations. Macrocidin‐induced changes in OJIP chlorophyll fluorescence parameters are consistent with inhibited electron transfer from QA to QB (potentially due to iron-binding), and uncoupling the light-harvesting complex (LHC) of photosystem II (PSII) from the reaction centre, leading to an increase in photoprotective xanthophylls. This latter impact was stronger in groundsel than chickpea or dandelion. The decrease in total carotenoid and carotenoid precursor content in macrocidin-treated plants could be explained by macrocidins’ magnesium-binding activity reducing the efficiency of 1‐deoxy‐D‐xylulose (DXP) reductoisomerase (DXR) and/or phytoene synthase (PSY). The putatively metal‐binding‐related modes of action of macrocidins occurred more rapidly than inhibition of PDS. These inter‐specific variations in, and diversity of, modes of action suggest that the risk of resistance developing to macrocidins is very low.

Evaluation of the functional roles of fungal endophytes of Phragmites australis from high saline and low saline habitats

Non-native Phragmites australis decreases biodiversity and produces dense stands in North America. We surveyed the endophyte communities in the stems, leaves and roots of collections of P. australis obtained from two sites with a low and high salt concentration to determine differences in endophyte composition and assess differences in functional roles of microbes in plants from both sites. We found differences in the abundance, richness and diversity of endophytes between the low saline collections (18 species distributed in phyla Ascomycota, Basidiomycota and Stramenopiles (Oomycota); from orders Dothideales, Pleosporales, Hypocreales, Eurotiales, Cantharellales and Pythiales; Shannon H = 2.639; Fisher alpha = 7.335) and high saline collections (15 species from phylum Ascomycota; belonging to orders Pleosporales, Hypocreales, Diaporthales, Xylariales and Dothideales; Shannon H = 2.289; Fisher alpha = 4.181). Peyronellaea glomerata, Phoma macrostoma and Alternaria tenuissima were species obtained from both sites. The high salt endophyte community showed higher resistance to zinc, mercury and salt stress compared to fungal species from the low salt site. These endophytes also showed a greater propensity for growth promotion of rice seedlings (a model species) under salt stress. The results of this study are consistent with the ‘habitat-adapted symbiosis hypothesis’ that holds that endophytic microbes may help plants adapt to extreme habitats. The capacity of P. australis to establish symbiotic relationships with diverse endophytic microbes that enhance its tolerance to abiotic stresses could be a factor that contributes to its invasiveness in saline environments. Targeting the symbiotic associates of P. australis could lead to more sustainable control of non-native P. australis.

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth.

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.

Impact of macrocidins, produced by Phoma macrostoma, on carotenoid profiles of plants

Phoma macrostoma (Montagne) is a bioherbicide that controls broadleaf weeds such as Canada thistle (Cirsium arvense L. (Scop.)) and dandelion (Taraxacum officinale Weber ex F.H. Wigg.), while leaving crop plants, such as wheat (Triticum aestivum L.) and pumpkin (Cucurbita L. spp), unharmed. The bioherbicidal activity of P. macrostoma is largely due to the production of phytotoxic secondary metabolites, macrocidins. While macrocidins produce photobleaching symptoms in susceptible plants, the mechanism(s) by which this occurs are not known. The hypothesis that macrocidins inhibit carotenoid biosynthesis was tested by comparing the carotenoid profiles and other physiological parameters of macrocidin-treated Canada thistle, dandelion, pumpkin and wheat with control plants and plants treated with the known carotenoid biosynthesis inhibitor, diflufenican. Only in susceptible plants, macrocidins induced photobleaching symptoms, lowered total chlorophyll content and reduced photosynthetic gas exchange while increasing the percentage of the carotenoid precursor phytoene. While diflufenican also led to chlorosis in dandelion and thistles, its impacts differed from those of macrocidins in that diflufenican-treated dandelion, thistle and pumpkin had increased total carotenoid and carotenoid precursor content compared to the control. Diflufenican-treated dandelion and thistle contained 88±3% and 94±2% phytoene, respectively. In contrast, macrocidins-treated dandelion and thistle (at the higher dose) contained 54±12% and 80±3% phytoene, respectively, while the controls contained only 4±0.5% and 10±1%. The β-carotene to lutein ratio in diflufenican-treated dandelion or thistles did not differ from that of the control, while this ratio was reduced in thistles treated with the higher dose of macrocidins. This paper provides the first proof that macrocidins impact the carotenoid biosynthesis by partially inhibiting the carotenoid biosynthetic enzyme phytoene desaturase (PDS) and at one or more other steps in carotenoid biogenesis.

Weed control spectrum and turfgrass tolerance to bioherbicide Phoma macrostoma

Phoma macrostoma is a bioherbicide being developed for selective weed control in turfgrass. The previous research with this product is limited to cool-season turfgrass systems with little information available on appropriate application rates or weed control spectrum for weeds common to warm-season turf. Studies were conducted to evaluate application rates and weed control spectrum of P. macrostoma on common dandelion (Taraxacum officinale G.H. Weber ex Wigg.), slender aster (Aster subulatus var. ligulatus Shinners), common mallow (Malva neglecta Wallr.), common purslane (Portulaca oleracea L.), California burclover (Medicago polymorpha L.), and annual sow thistle (Sonchus oleraceus L.). Although most weeds exhibited susceptibility to P. macrostoma, control differed by weed species and study. P. macrostoma had the greatest efficacy on dandelion, but provided little efficacy on common purslane and common mallow. Warm-season turfgrass tolerance was also evaluated across five commonly used warm-season turfgrass species. No turfgrass injury was observed at any rate on any species; however, weed control data suggest that P. macrostoma may not offer effective broad-spectrum weed control similar to common synthetic herbicides, when applied at the rates used in this study.

Soil sickness of peanuts is attributable to modifications in soil microbes induced by peanut root exudates rather than to direct allelopathy

The quantity and quality of peanut yields are seriously compromised by consecutive monoculture in the subtropical regions of China. Root exudates, which represent a growth regulator in peanut–soil feedback processes, play a principal role in soil sickness. The growth inhibition of a species in an in vitro bioassay enriched with root exudates and allelochemicals is commonly viewed as evidence of an allelopathic interaction. However, for some of these putative examples of allelopathy, the results have not been verified in more natural settings with plants continuously growing in soil. In this study, the phenolic acids in peanut root exudates, their retention characteristics in an Udic Ferrosol, and their effects on rhizosphere soil microbial communities and peanut seedling growth were studied. Phenolic acids from peanut root exudates were quickly metabolized by soil microorganisms and did not accumulate to high levels. The peanut root exudates selectively inhibited or stimulated certain communal bacterial and fungal species, with decreases in the relative abundance of the bacterial taxa Gelria glutamica, Mitsuaria chitosanitabida, and Burkholderia soli and the fungal taxa Mortierella sp. and Geminibasidium hirsutum and increases in the relative abundance of the bacterial taxon Desulfotomaculum ruminis and the fungal taxa Fusarium oxysporum, Bionectria ochroleuca and Phoma macrostoma. The experimental application of phenolic acids to non-sterile and sterile soil revealed that the poor performance of the peanut plants was attributed to changes in the soil microbial communities promoted by phenolic acids. These results suggest that pathogenic fungal accumulation at the expense of such beneficial microorganisms as plant growth promoting rhizobacteria, mycorrhizal fungi induced by root exudates, rather than direct autotoxicity induced by root exudates, might represent the principal cause underlying the soil sickness associated with peanut plants. We hope that our study will motivate researchers to integrate the role of soil microbial communities in allelopathic research, such that their observed significance in soil sickness during continuous monocropping of fields can be further explored.

Pyromorphite formation in a fungal biofilm community growing on lead metal

Lead is a priority pollutant, and lead metal is widely found in the environment as a waterproofing structural component in roofing, fence post covers, venting and flashing, as well as in industrial and urban waste. However, little is known of microbial interactions with metallic lead. The objective of this research was to investigate fungal roles in transformations of lead in a surface biofilm community growing on lead sheeting. The lead surface was found to support a diverse fungal community with several members, such as Aureobasidum pullulans, Phoma macrostoma, Penicillium sp. and Botryotinia fuckeliana, probably originating from adjacent phylloplane communities. Many fungal isolates showed tolerance to lead compounds in growth inhibition assays and were able to mediate production of lead-containing secondary minerals in the presence of metallic lead. These exhibited widely differing morphologies to the lead-containing secondary minerals produced under abiotic conditions. The presence of pyromorphite (Pb5 (PO4 )3 Cl) (approximately 50 wt%) was detected in the lead sheet biofilm, and we speculate that animal (bird) faeces could be a significant source of phosphorus in this location. Pyromorphite formation represents biomineralization of mobile lead species into a very stable form, and this research provides the first demonstration of its occurrence in the natural environment.