Transformation of the Fungal Soybean Pathogen Cercospora kikuchii with the Selectable Marker bar

An improved transformation protocol, utilizing selection for resistance to the herbicide bialaphos, has been developed for the plant pathogenic fungus Cercospora kikuchii. Stable, bialaphos-resistant transformants are recovered at frequencies eight times higher than those achieved with the previous system that was based on selection for benomyl resistance. In addition to C. kikuchii, this improved method can also be used to transform other species of Cercospora.     

Relationship between Late Soybean Diseases Complex and Rain in Determining Grain Yield Responses to Fungicide Applications

Wide distribution of soybean monoculture associated with no tillage has contributed to enhance damages caused by late diseases complex (LDC) in Argentina. LDC is a complex of diseases where Septoria glycines and Cercospora kikuchii are regarded as the major problem. Even though the use of foliar fungicides has increased, there is no rational and economic guide for their use. This is the main reason why the response to foliar fungicide applications is unpredictable. One of the main factors that contribute to the development of LDC is rainfall. The objective of this study was to evaluate the impact of rainfall during several growing seasons and different soybean growth stages on LDC severity and yield. We carried out 18 field experiments during three growing seasons (2004–2006) at several locations in the Argentine Pampas Region, to examine the relationship between rain and yield response to single fungicide applications (quinone outside inhibitors and demethylation inhibitors) at growing stages R3 and R5. The strongest associations (R² = 0.81–0.84; P < 0.001) were observed between accumulated rainfall from R3 to R5 and yield response to fungicides applied in R3 or R5. Our results suggest that a minimum of 65–90 mm rainfall during R3–R5 is required to justify fungicide application, with high probability that the use of fungicide will increase soybean yield as a consequence of disease control. These findings could lead to a simple model, useful as decision support system for use in planning and scheduling spray applications for LDC management in soybean crops.     

More Cercospora Species Infect Soybeans across the Americas than Meets the Eye

Diseases of soybean caused by Cercospora spp. are endemic throughout the world’s soybean production regions. Species diversity in the genus Cercospora has been underestimated due to overdependence on morphological characteristics, symptoms, and host associations. Currently, only two species (Cercospora kikuchii and C. sojina) are recognized to infect soybean; C. kikuchii causes Cercospora leaf blight (CLB) and purple seed stain (PSS), whereas C. sojina causes frogeye leaf spot. To assess cryptic speciation among pathogens causing CLB and PSS, phylogenetic and phylogeographic analyses were performed with isolates from the top three soybean producing countries (USA, Brazil, and Argentina; collectively accounting for ~80% of global production). Eight nuclear genes and one mitochondrial gene were partially sequenced and analyzed. Additionally, amino acid substitutions conferring fungicide resistance were surveyed, and the production of cercosporin (a polyketide toxin produced by many Cercospora spp.) was assessed. From these analyses, the long-held assumption of C. kikuchii as the single causal agent of CLB and PSS was rejected experimentally. Four cercosporin-producing lineages were uncovered with origins (about 1 Mya) predicted to predate agriculture. Some of the Cercospora spp. newly associated with CLB and PSS appear to represent undescribed species; others were not previously reported to infect soybeans. Lineage 1, which contained the ex-type strain of C. kikuchii, was monophyletic and occurred in Argentina and Brazil. In contrast, lineages 2 and 3 were polyphyletic and contained wide-host range species complexes. Lineage 4 was monophyletic, thrived in Argentina and the USA, and included the generalist Cercospora cf. flagellaris. Interlineage recombination was detected, along with a high frequency of mutations linked to fungicide resistance in lineages 2 and 3. These findings point to cryptic Cercospora species as underappreciated global considerations for soybean production and phytosanitary vigilance, and urge a reassessment of host-specificity as a diagnostic tool for Cercospora.     

Genetic Diversity of <Emphasis Type="Italic">Cercospora kikuchii</Emphasis> Isolates From Soybean Cultured in Argentina as Revealed by Molecular Markers and Cercosporin Production

Leaf blight and purple seed, caused by the fungal pathogen Cercospora kikuchii (Matsumoto & Tomoyasu) M. W. Gardner are very important diseases of soybean (Glycine max L. Merr.) in Argentina. The aims of this work were: (a) to confirm and to assess the genetic variability among C. kikuchii isolates collected from different soybean growing areas in Santa Fe province using inter simple sequence repeats (ISSR) markers and sequence information from the internal transcribed spacer (ITS) region of rDNA and (b) to analyze the cercosporin production of the regional C. kikuchi isolates in order to assess whether there was any relationship between the molecular profiles and the toxin production. Isolates from different regions in Santa Fe province were studied. The sequence of the ITS regions showed high similarity (99–100%) to the GenBank sequences of C. kikuchii BRCK179 (accession number AY633838). The ISSR markers clustered all the isolates into many groups and cercosporin content was highly variable among isolates. No relationship was observed between ITS region, ISSR groups and origin or cercosporin content. The high degree of genetic variability and cercosporin production among isolates compared in this study characterizes a diverse population of C. kikuchii in the region.     

In vitro evaluation of the efficacy of plant and callus extracts of Cardiospermum halicacabum L. on important phytopathogenic fungi

The aqueous and different solvent extracts viz., petroleum ether, chloroform, methanol and ethanol extracts of leaf and leaf derived callus of Cardiospermum halicacabum L. (Sapindaceae) at different concentrations were screened in vitro for antifungal activity by the poisoned food technique against a wide array of seed-borne phytopathogenic fungi. The test organisms include Aspergillus flavus, A. niger, Curvularia lunata, Drechslera halodes, Fusarium moniliforme, F. solani, and F. oxysporum, which are frequently associated with sorghum [Sorghum bicolor (L.) Moench], maize (Zea mays L.) and paddy (Oryza sativa L.) seeds. Aqueous leaf extracts of C. halicacabum showed significant inhibition was observed at 50% concentration particularly in Aspergillus species. With regard to the comparative efficiency of leaf and leaf derived callus extracts, aqueous leaf extract was found to be more effective than callus extract. Among the different solvent extracts, it was observed that at 3000 ppm concentration methanol extract of C. halicacabum leaf recorded the highest degree of activity and percentage inhibition was more, but in petroleum ether and chloroform extracts did not show any significant activity. C. halicacabum leaf derived callus at 3000 ppm methanol extract showed significant antifungal activity on Fusarium species. Leaf of C. halicacabum aqueous and methanol extract showed significant antifungal activity against all the tested fungi. C. halicacabum has significant medicinal value, hence the results of the present investigation indicate that it could be exploited in the management of seed-borne pathogenic fungi.     

Soybean Toxin (SBTX) Impairs Fungal Growth by Interfering with Molecular Transport,Carbohydrate/Amino Acid Metabolism and Drug/Stress Responses

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.     

Antibacterial and antifungal activities of teucrium royleanum (Labiatea)

The crude methanolic extract and subsequent fractions of Teucrium royleanum (Labiatea) were screened for antibacterial and antifungal activities. Against tested pathogens, crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities. Highest antibacterial activity was displayed by the ethyl acetate fraction against S. typhi (100%), against E.coli (76.7%) and against P. aerugenosa (70.8%) followed by the chloroform fraction against S. typhi (85.7%). Similarly, the crude extract and its subsequent fractions showed mild to excellent activities in the antifungal bioassay with maximum antifungal activity against M. canis (87%) by the chloroform fraction followed by the ethyl acetate (71%) and n-butanol (70%) fractions.     

A phylogenetic study of some <Emphasis Type="Italic">Septoria</Emphasis> species pathogenic to Asteraceae based on ITS ribosomal DNA sequences

The phylogenetic relationships of the following 14 Septoria species pathogenic to Asteraceae were inferred from sequence analyses of the 5.8 S gene of nuclear rDNA and flanking internal transcribed spacers: Septoria atropurpurea, S. astericola, S. calendulae, S. erigerontis, S. gerberae, S. helianthi, S. helianthicola, S. lactucae, S. leucanthemi, S. obesa, S. schnabliana, S. senecionis, S. socia, and S. taraxaci. Fifteen additional Septoria species originating from other host families, eight of which have a known Mycosphaerella teleomorph, were included in the alignment. Also included were selected strains of Cercospora, Pseudocercospora (including Paracercospora), Ramularia, and several other anamorph genera linked to Mycosphaerella available from previous studies. The analyses indicate that the Septoria species from Asteraceae are all closely related, and share an evolutionary history within the Mycosphaerellaceae, grouping amongst miscellaneous Mycosphaerella species, Cercospora and Pseudocercospora spp. Septoria socia is relatively distant from the other Septoria on Asteraceae, and the data suggest that it is more closely related to the maple pathogen S. aceris (tel. M. latebrosa) and poplar pathogen S. populicola (tel. M. populicola). Differences in the ITS sequences indicate that S. socia and S. leucanthemi, which have very similar phenotypes and have been considered to be conspecific in the past, are specifically distinct.     

In vitro evaluation of the antifungal activity of Allium sativum bulb extract against Trichophyton rubrum,a human skin pathogen

The aqueous extract of Allium sativum bulbs showed an antifungal effect against the fungal skin pathogen, Trichophyton rubrum, isolated from infected patients. For a given concentration (200 mg of bulbs/1 ml), the volume of the aqueous garlic extract loaded on to the discs, and the diameter of the inhibitory zone formed around the disc on the fungal lawn showed positive correlation. Extract-included microbial assay confirmed the antifungal effect of Allium sativum. The extract was not heat stable, it lost its antifungal property above 60 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Herbicides and Oat Amendment on Soybean Disease Development and Seed Quality

The effects of four herbicides and hand weeding and no weeding on soybean disease development and seed quality were studied in the field. Chloramben, dinoseb, fluchloralin, and trifluralin were preplant incorporated at recommended rates. In addition, the effect of an in-furrow oat (Avena sativa L.) amendment used as an inoculum carrier was studied in all treatments. Compared with the hand-weeded control, yield, incidence of Septoria leafspot (Septoria glycines), and the recovery of Phomopsis spp. from pod and stem tissues were reduced in the week-infested control and dinosebtreated plots. Recovery of Alternaria spp. was greater from crown tissues of plants grown in chloramben- and fluchloralin-treated plots compared with, either the weed-infested or hand-weeded control. Septoria leafspot ratings and recovery of Phomopsis spp. from various plant tissues were higher in plants from hand-weeded plots compared with weed-infested plots, whereas the opposite was truefor, the recovery of Fusarium spp. Chloramben-treated plots were high-yielding and had the best weed control of all herbicide treatments. Seed quality was not affected by herbicide treatment. Plants from furrows with autoclaved oats had a lower incidence of Septoria glycines, a greater recovery of Fusarium spp. from pod and stem pieces, and produced seed with lower quality than plants from rows with no oat amendment.     

Antimicrobial bioassay of colchicum luteum baker

The methanolic extract of the corms of Colchicum luteum Baker (Liliaceae) and its subsequent fractions in different solvent systems were screened for antibacterial and antifungal activities. The crude extract and all the fractions demonstrated moderate to excellent antifungal activities against tested pathogens in antifungal bioassay. Excellent antifungal activity was shown against trichophyton longifusus, up to 75%, and microsporum canis, up to 85%, while the crude extract and subsequent fractions showed mild to moderate activities in an antibacterial bioassay with maximum antibacterial activity 58% against Bacillus subtilis.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Interaction between pathogenic and saprobic fungi isolated from soybean roots and seeds

A variety of interactions was recorded in culture between 11 saprobic fungi isolated from soybean (Glycine max) roots and seeds and the soybean pathogens Cercospora sojina, Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae, and Septoria glycines. The most active saprobes were Aspergillus terreus, Chaetomium cupreum, Epicoccum nigrum, Gliocladium roseum, Myrothecium roridum, Penicillium thomii, and Trichothecium roseum. Hyphal lysis of several fungal pathogens by Acremonium sp., C. cupreum and P. thomii was recorded perhaps because of parasitism by G. roseum and T. roseum. In greenhouse studies, seeds coated with G. roseum, P. thomii, and T. harzianum emerged significantly (P=0.05) more than those coated with A. terreus and the control. In field studies, seeds coated with a conidial suspension of A. terreus, G. roseum, P. thomii or Trichoderma harzianum produced a significantly greater stand than the control. The area of cotyledons covered with lesions caused by C. truncatum was significantly less on seeds coated with G. roseum, P. thomii and T. harzianum than the control.     

Exploring the antidiabetic and anti-obesity properties of Samanea saman through in vitro and in vivo approaches

In recent years, diabetes and obesity have become a major problem in global health care because of changes in lifestyle, food habits, and age-related metabolic disorders. Diabetes mellitus is one of the most common diseases, affecting millions of people worldwide. Currently, herbal drugs are used to control obesity and diabetes. The present study investigates the anti-obesity, antidiabetic, and antioxidant activities of Samanea saman leaf extract. A methanolic extract of S. saman leaves was prepared by a maceration method. The S. saman leaf extract was studied for its inhibitory effect on glucose utilization using specific in vitro procedures to analyze its antioxidant, anti-obesity, and antidiabetic activities via different assays, such as α-amylase and α-glucosidase inhibition assay, glucose uptake by yeast cells, nonenzymatic glycosylation assay followed by glucose diffusion assay. The outcome of the study showed that the methanolic extract strongly inhibited the pancreatic lipase, α-amylase, and glucosidase activities, compared with the standard drug. The results showed that the extract possessed considerable antioxidant and antidiabetic activities, and further studies are needed to confirm the results using an in vivo model. Thus, it is proposed that S. saman can be used as a therapeutic agent.     

Antibacterial and antifungal activities of andrachne cordifolia muell

The crude methanolic extract of Andrachne cordifolia Muell. (Euphorbiaceae) and its various fractions in different solvent systems (chloroform, ethyl acetate and n- butanol) were screened for antibacterial and antifungal activities. Crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities against the tested pathogens. Highest antibacterial activity was displayed by both chloroform and ethyl acetate fractions (100%) followed by the crude extract (68%) against Salmonella typhi. Similarly, crude extract and its subsequent fractions showed mild to excellent activities in antifungal bioassay with maximum (76%) antifungal activity against Microsporum canis by the chloroform fraction followed by the crude extract (65%).     

Fungitoxic properties of selected South African plant species against plant pathogens of economic importance in agriculture

Thirty‐nine plant species, representing 20 families from the subclasses Rosidae, Asteridae, Commelinidae and Liliidae, were collected from the Blyde River Canyon Nature Reserve, Mpumalanga, South Africa. Crude extracts were prepared and bioassayed, at equal concentrations, for their antifungal potential by determining the inhibitory effects on the mycelial growth of seven economically important plant pathogenic fungi. Statistically significant differences between plants and plant parts were observed as well as the resistance of different fungi to treatment with different plant extracts. The most significant mycelial growth inhibition was obtained with extracts from two species of the subclass Liliidae, namely Aristea ecklonii and Agapanthus inapertus. The crude extract of A. ecklonii performed best of all extracts as it totally inhibited the mycelial growth of all seven of the plant pathogenic test organisms and outperformed the inhibition by a broad spectrum synthetic fungicide (carbendazim/difenoconazole). Crude extracts of A. inapertus showed complete inhibition of four and strong inhibition of the remaining three plant pathogenic fungi. Although not as efficient as the previous two species, the extract of Anisopappus junodii, from the subclass Asteridae, also showed promising antifungal activity by completely inhibiting mycelial radial growth of two and strongly inhibiting that of the remaining five fungi.     

Genetic Transformation System for the Fungal Soybean Pathogen Cercospora kikuchii

An altered β-tubulin gene that confers resistance to the fungicide benomyl was isolated from a genomic library of a UV-induced mutant of Cercospora kikuchii and used as a selectable marker for transformation. The level of benomyl resistance conferred to the transformants was at least 150-fold greater than the intrinsic resistance of the C. kikuchii recipient protoplasts. In the majority of cases, the tubulin fragment was integrated at the native β-tubulin locus, apparently by gene replacement or gene conversion. The frequency of transformation ranged from 0.2 to 6 transformants per μg of DNA, depending on the recipient strain. Transformation with linearized plasmid resulted in a higher frequency, without changing the type of integration event. Transformants were phenotypically stable after eight consecutive transfers on medium without benomyl. This is the first report of a genetic transformation system for a Cercospora species.     

In vitro antimicrobial activity of zimmu ( Allium sativum L. ‘ Allium cepa L.) leaf extract

The fungitoxic effect of various medicinal plants belonging to different families was evaluated in vitro on Rhizoctonia solani, the rice sheath blight pathogen. Of the various plant extracts, the leaf extract of zimmu (Allium cepa × Allium sativum) showed the maximum antifungal activity against R. solani and recorded an inhibition zone of 12?mm. The leaf extract of zimmu was also effective in inhibiting the growth of other agronomically important fungal and bacterial pathogens viz., Aspergillus flavus, Curvularia lunata, Alternaria solani, Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. malvacearum and Xanthomonas axonopodis pv. citri. The antimicrobial compound was dissoluble in methanol and the methanolic extract showed the absorption maxima at 210?nm and 230?nm. Phenolic compounds were present in greater amounts in methanol extract of zimmu. TLC analysis showed the appearance of two blue spots at R _f ?=?0.65 and R _f ?=?0.90. The compounds eluted at R _f ?=?0.65 and R _f ?=?0.90 by preparative TLC exhibited strong antifungal activity against R. solani.     

Factors affecting incidence and severity of leaf spot disease on lettuce caused by Septoria birgitae

In the 1990s during wet seasons a new disease causing brown leaf spots on lettuce (Lactuca sativa) was found for the first time in many lettuce‐growing areas of Austria and Germany. The causal agent, a new pathogenic species called Septoria birgitae, may be responsible for total crop loss. To study how temperature, inoculum density and leaf wetness period influence disease incidence and severity of leaf spot on lettuce caused by S. birgitae, we carried out in vivo experiments in growth chambers and in the field. Additionally, we evaluated the relevance of infected plant debris acting as a primary inoculum source in soil for subsequent crops. S. birgitae produces spores over a wide temperature range between 5°C and 30°C, and can infect plants at temperatures between 10°C and 30°C, with an optimum between 20°C and 30°C. Spores of S. birgitae at a density of at least 10³ conidia mL^–1 are essential for disease outbreak on lettuce. Because leaf wetness is crucial for releasing conidia from pycnidia, we studied the impact of leaf wetness duration on disease development under various temperature conditions. For relevant leaf spot disease development on lettuce in vivo, a leaf wetness duration of at least 24 h and temperatures higher than 10°C were necessary. Leaf spot disease development in the field required several leaf wetness periods longer than 20 h at approximately 15°C at the beginning of crop cultivation. Incorporating S. birgitae infected plant debris in soil as a primary inoculum was not relevant for leaf spot disease outbreak in the next year. However, in cases of continuous cropping of lettuce on the same field and in the same season, Septoria‐infected lettuce debris may become more relevant.     

Screening the antimicrobial potential of twelve medicinal plants against venereal diseases causing pathogens

The antimicrobial potential of selected ethnomedicinal plants in traditional healers of Silent valley, Palakkad district of Kerala, India against venereal diseases causing pathogens. Twelve medicinal plants and their parts were collected from the various places of Silent Valley, Kerala, India. These plant parts were dried and mixed with different kinds of solvents respectively hexane, chloroform, methanol and water. In this study, six microbial strains were selected, in which five bacterial and a fungal strain. Among the bacterial strains, two strains were gram positive and three strains for gram negative bacteria. Totally, twelve medicinal plant parts mixed with various solvents were treated against the selected pathogenic organisms. Among them, methanolic extract of A. occidentate, C. indica, H. rosa-sinensis and M. oleifera exhibited excellent antibacterial activity than other parts of plants and standard drugs. As well as, methanolic extract of H. rosa-sinensis showed good antifungal activity against C. albicans. While,the least inhibition was noted with aqueous extract of C. indica against S. aureus. The MIC ranges from 0.78μg/ml to 50μg/ml and MBC/MFC 1.52μg/ml to 50μg/ml. The methanolic flower extract of H. rosa-sinensis has showed effective zone of inhibition against all the pathogens, particularly N. gonorrhoeae (30 mm) and C. albicans (26mm), than other extracts and standard drugs. Therefore, we concluded that, flower extract had potential therapeutic activity against venereal diseases. This research will be helpful to discover the new therapeutic drug molecule in Pharmaceutical sectors.