Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).     

Medium optimization for the production of the secondary metabolite squalestatin S1 by a Phoma sp. combining orthogonal design and response surface methodology

In the present work, a combined statistical approach of orthogonal design (L₂₇(3¹³)), response surface techniques and polynomial regression were applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a fungus (a Phoma species). Optimal conditions for maximal titres and productivity were determined based on 13 parameters at three different levels. Initially, a screening design methodology was used to evaluate the process variables, which were relevant to S1 titre and the response surfaces applied to find optimal regions for production. The sources of carbon and concentration, and their interactions with oily precursors were statistically significant factors. The combined orthogonal design and response surface methodology predicted optimal conditions for of 273 mg l⁻¹ of squalestatin S1. Confirmatory experiments of the optimal medium composition produced titres of 434 mg l⁻¹ in a 5-day fermentation at 25 °C. This represented a 60% improvement in the maximum titre predicted, and a two-fold higher productivity when compared with reported S1 yields of various fungal species. This combined statistical approach enables rapid identification and integration of key medium parameters for optimising secondary metabolite production and could be very useful in pharmaceutical screening programmes.     

Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae)

Seeds of Opuntia spp. have physiological dormancy; they need a period of after-ripening to break dormancy, and the embryos have low growth potential. We evaluated the combined effects of seed age and presence of fungi on the testa on germination of Opuntia streptacantha, an abundant species in the Chihuahuan Desert (Mexico), assuming that older seeds have broken seed dormancy and fungi can reduce mechanical resistance to germination. In a preliminary experiment, we found no germination of 9-year-old (1998) and freshly collected (2007) seeds. However, we obtained 67% and 27% germination from 9-year-old and fresh non-sterilized seeds, respectively, and found fungi growing on the testa of all germinated seeds. Two fungal strains were isolated and identified using ribosomal internal transcribed spacer (ITS) sequence analysis: Penicillium chrysogenum and Phoma sp. In a second experiment, we inoculated seeds with strains of P. chrysogenum and Phoma sp., as well as Trichoderma koningii and binucleate Rhizoctonia (Gto17S2), to evaluate their ability to break seed dormancy. Seeds inoculated with P. chrysogenum, Phoma sp. and T. koningii had higher germination than controls for both seed ages, but germination was higher in older seeds. Scanning electron microscopy showed that these fungi eroded the funiculus, reducing its resistance. Binucleate Rhizoctonia did not lead to germination and controls had almost no germination. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. streptacantha, and that the effect of fungi on seeds is species-specific.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Advances in Taxonomy of Genus Phoma: Polyphyletic Nature and Role of Phenotypic Traits and Molecular Systematics

Phoma is a highly polyphyletic genus with its unclear species boundaries. The conventional system of identification is functional but it has its limitations. Besides morphological studies, chemotaxonomy, secondary metabolite and protein profiling have been assessed for the classification and identification of these fungi. Molecular datasets have provided a better outlook towards the phylogenetic and evolutionary trends of Phoma. Molecular markers such as ITS-rDNA, tubulin, actin, translation elongation factor have been widely used by the taxonomists to demarcate species. However, outcomes gained up till now represent preliminary step towards the study of Phoma systematics and a combined approach would be beneficial in the understanding of this polyphyletic group members. Lately, on the base of molecular phylogeny of the type species of the seven Phoma sections a new teleomorph family, Didymellaceae has been established, besides the Phaeosphaeriaceae related to sect. Paraphoma anamorphs, and the Leptosphaeriaceae to sect. Heterospora anamorphs. The estimated ratio is about 70 % of the recognized Phoma-like species can be associated with the Didymellaceae ascomycetous family.     

Formulation of phytotoxins from Phoma sp. FGCC#18 for the management of Parthenium hysterophorus L.

Biological control is a relatively cheap and most feasible long-term approach for controlling deadly weeds. Parthenium hysterophorus L. is a pernicious weed, imposing deleterious economic effects on humans and livestock. Microbial products thus offer a non-chemical alternative for controlling a noxious, invasive and pernicious weed like Parthenium. Phoma is a well-known phytopathogen responsible for many diseases in plants and is known to produce an array of bioactive extracellular toxic compounds, i.e. agribiologicals. Before application these have to be effectively formulated to enhance their absorption and translocation within the foliar tissues. Adjuvants are formulatives of microbial bioherbicides which ensure maximum herbicidal performance. Research on adjuvant and formulation technology for ecofriendly agrochemicals has advanced rapidly in recent years. Phytotoxins from Phoma sp. FGCC#18 have been tested for their compatibility with different formulants. Phytotoxicity damage was studied by seedling and detached leaf bioassays after different hours post-treatment. Tween 80 at 0.5% was found to be the best formulant, while the combination of Tween 80 and coconut oil at 0.5% exhibited maximum damage to Parthenium seedlings and detached leaves.     

The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.     

STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000     

Factors affecting development of phoma canker (Leptosphaeria maculans) on stems of winter oilseed rape (Brassica napus) in southern England

In winter oilseed rape experiments at Rothamsted in 1997/98 (cvs Lipton and Capitol), 1998/99 (cv. Apex) and 1999/2000 (cvs Apex, Lipton and Capitol), development of crown canker and phoma stem lesions in spring was related to development of phoma leaf spot in the previous autumn/winter. There were differences in thermal time (degree‐days) from the first appearance of phoma leaf spot (autumn) to the first appearance of crown canker (spring) between cultivars (cvs Lipton and Capitol, 1220–1240; cv. Apex, 1120–1140 degree‐days) but not between growing seasons. In 1998/99 and 1999/2000, fungicide (November) treatment delayed the start of crown canker development in the spring but did not affect the rate of increase in severity. In 1997/98, fungicide treatments did not delay the appearance of crown canker but decreased the rate of increase in crown canker severity. In all three seasons, fungicide treatments generally decreased the proportions of plants at harvest with crown canker severity scores 3 or 4 and increased the proportions with scores 0 or 1. There were differences between seasons in the distributions of crown canker severity scores at harvest. The severity of both crown canker and phoma stem lesions increased linearly with accumulated degree‐days in plots with or without fungicide treatment in 1997/98 (cv. Lipton), 1998/99 (cv. Apex) and 1999/2000 (cv. Apex). Regressions showed that severity of crown canker at harvest in July was related to severity in the spring in 1997/98 (early June, cv. Lipton), 1998/99 and 1999/2000 (April, cv. Apex).