Development of Seed Immunoblot Binding Assay for Detection of Karnal bunt (Tilletia indica) of Wheat

Polyclonal antisera were produced in albino white rabbits against intact teliospores of Karnal bunt (Tilletia indica). The Immunoprobe generated was used for the development of Immunoblot binding assay for detecting Karnal bunt (KB) infections in wheat seed samples. The antiserum reacted strongly with intact teliospores of T. indica, Pantnagar isolate in agglutination reaction. Wheat grains with different grades of infection could be readily detected by Seed Immunoblot Binding Assay (SIBA). Karnal bunt infected wheat seeds when kept for vigour testing on nitrocellulose paper, formed a coloured imprint after the paper was immunoprocessed. The SIBA would not only be a better Indication of teliospores load on seed but also quality of seed in terms of vigour. This method is expected to be useful in routine monitoring of wheat lots for the presence of KB teliospores.     

A protein-nucleic acid complex in teliospores of Ustilago maydis

The ribonucleic acids extracted from nongerminated teliospores of Ustilago maydis showed the spectrum similar to histone-DNA complex with a high 230/260 mμ ratio. The chemical analyses of the RNA preparations revealed that the spectral abnormality was due to the complexing of RNA with a protein. On the other hand RNA prepared from the isolated ribosomes of spores exhibited a normal spectrum and low protein content relative to RNA fraction. These observations show that an unusual protein is present in germinated spores which complexes with RNA and disappears as germination occurs.     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific literature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific luterature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

New geographic distribution records and assumed microcyclic life cycle of Triphragmiopsis jeffersoniae (Pucciniales)

Triphragmiopsis jeffersoniae was found on Jeffersonia dubia in Sapporo, Japan, in 2003. This was the first geographic distribution record of the fungus out of its native distribution range in continental Far East Asia. This fungus was found also in Nikko, Japan, in 2011. Triphragmiopsis jeffersoniae formed Aecidium-type sori and tripartite teliospores subtended by a pedicel. In the early spring, the fungus formed telia with or without Aecidium-type sori on petioles of emerging host leaves. Subsequently clusters of Aecidium-type sori were formed on the abaxial leaf surface. These sori did not repeat; but they were soon densely surrounded by telia. Both the Aecidium-type sori and telia were densely crowded and no small isolated telia were formed. The assumption that the spores from Aecidium-type sori do not have infective ability was confirmed by inoculation experiments and microscopic observations for the Japanese materials. The Aecidium-type spore germlings failed to invade the Jeffersonia leaf either directly or through a stoma under the experimental conditions. Thus, T. jeffersoniae was assumed to have microcyclic life cycle, comprising the functional teliospores and the non-functional Aecidium-type aeciospores.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

Melampsora salicis-sinicae (Melampsoraceae,Pucciniales), a new rust fungus found on willows in China

Based on morphological characteristics and molecular phylogeny, a rust fungus on Salix hsinganica, S. sinica, S. starkeana, S. wallichiana and S. xerophila in China is described as a new species, Melampsora salicis-sinicae. This new species morphologically resembles M. capraearum and M. epiphylla in having subcuticular teliospores with a thickened apical wall, but it differs from these two species in its amphigenous telia. In addition, this new species differs from M. capraearum in its slender teliospores and thinner apical thickness of teliospores, and it also differs from M. epiphylla in the dimension of urediniospores, the position of teliospores and the apical thickness of teliospores. Phylogenetic analyses using partial sequences of the D1/D2 region of nuclear large subunit and the internal transcribed spacer (ITS1 and ITS2) regions including 5.8S of ribosomal RNA gene and the translation elongation factor 1 alpha (EF-1α) gene revealed that this rust fungus was distinct from its morphologically similar species, M. capraearum and M. epiphylla.     

Fixation of Dormant Tilletia Teliospores for thin Sectioning

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall- Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20, and 25 μm). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructurc and histochemistry of dormant spores with those of germinated spores.     

Karnal bunt: A minor disease that is now a threat to wheat

Karnal bunt of wheat, caused byNeovossia indica (Mitra) Mundkur, was first reported in 1931 from Northwestern India in experimental plantings. For many years it was a minor disease found only in Northwestern India. During the 1969–70 crop season it was unusually widespread in Northwest India and since 1974–75 Karnal bunt has been distributed throughout Northern India from West Bengal to the western border. Seed lots with more than 50% of the kernels infected have been collected from farm threshing sites. The disease is now established in Afghanistan, Iraq, Pakistan and Mexico and has been intercepted in India in wheat seed that was shipped from Lebanon and Syria. The increased geographic distribution of Karnal bunt may be due to the development and wide distribution of wheat cultivars that were more susceptible than the older wheats that were grown in India prior to 1969–70. Resistance to Karnal bunt has been reported in several Indian wheats but most cultivars are susceptible. Hexachlorobenzene and cyano (methylmercuric) guanidine each prevented germination of teliospores ofN. indica in tests in Mexico.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

<Emphasis Type="Italic">Phragmidium</Emphasis> species parasitizing species of Rosaceae in Tibet,China, with descriptions of three new species

Phragmidum spp. are rust fungi that are serious pathogens of plants in the Rosaceae. We characterized 15 Phragmidium species from Tibet, including 12 previously described and 3 new species. All the taxa, including the three new species (Ph. chayuensis, Ph. cibanum, Ph. zangdongii) are described and illustrated based on morphological characteristics. These taxa have multi-celled teliospores with firm pedicels. Phragmidium chayuensis is characterized by (5)7–9-celled teliospores with short papillae. Phragmidium cibanum is characterized by smooth teliospores that are distinctly constricted at the septa. Phragmidium zangdongii is characterized by large uredinia and 11–13-celled teliospores. Five types of urediniospore-surface structures were identified based on the gross shape of ornamentations and their distribution on the urediniospore wall. Molecular sequence data from the LSU rDNA analysis showed that the new taxa formed distinct clades independent from previously recorded species represented by LSU rDNA sequence data. The phylogenetic tree indicated that Phragmidium species are highly host specific.     

Teliospore development of Narasimhania alismatis in artificial culture

Development of teliospores interlaced by dermata in clathroid spore balls by the aquatic smut Narasimhania alismatis Pavgi & Thirumalachar was achieved in artificial culture. The spores are morphologically similar to the spore balls in the host (Alisma reniforme D. Don.) leaf. The teliospore development is not conditioned by any nutritrional factor(s) and occurs also in the rich marshy soil,adding another source of primary inoculum     

Einsatz von V. lecanii als biologisches Schädlingsbekämpfungsmittel gegen den Bohnenrostpilz U. appendiculatus var. appendiculatus im Feld und im Gewächshaus

The use of Verticillium lecanii as a biological control agent against the bean rust fungus Uromyces appendiculatus var. appendiculatus in the field and in the glasshouse The deuteromycete V. lecanii parasites uredo- and teliospores of the bean-rust-fungus U. appendiculatus var. appendiculatus. We investigated the conditions for the use of the hyperparasite as biological control agent in the field and in glasshouses. The growth rate of the hyperparasite was 0,3 cm per day at 25 °C. Under suitable conditions in the lab (25 °C, 100 % r. h.) it took about 20 days to invade 100 % of uredospores and 65 % of teliospores. We failed to prevent the spread of bean-rust-fungus spores in the field, but we succeeded in the glasshouse by 68 %, compared to the untreated controls, using the hyperparasite V. lecanii as biological control agent.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

Source strength of wheat pathogens during combine harvest

Many fungal pathogens of plants are dispersedaerially long distances and by this meansestablish disease foci and redistribute races.The goal of this research was to measure thesource strength of wheat pathogens duringharvest. Two fields in North Dakota wereplanted with spring wheat cultivar 2375 andcombined from windrows. A Rotorod model 20 airsampler attached to a helium balloon was liftedto 30 m and activated when the debris cloudfrom the combine passed the sampler location.The sampler was positioned 100 to 200 mdownwind from the field edge and 100 m upwindof the combine in field one. In this field,concentrations of Puccinia triticinauredospores were 1663 spores/m³ downwindand 19 spores/m³ upwind; also,Cochliobolus sativus conidia were 732spores/m³ downwind and 32 spores/m³upwind. In field two, downwind samples weretaken at heights of 6 m and 30 m on the fieldedge. In this field P. triticina sporeconcentration was 840 spores/m³ downwind.Also in field two, conidia of Pyrenophoratritici-repentis and teliospores ofUstilago sp. were each observed atconcentrations of 9 spores/m³. Many sporesappeared desiccated, but C. sativusspores germinated after 24 h in a moistchamber. Spore concentrations at the fieldmargin meant a minimal estimate of 10 billionspores were dispersed from the field per hourof combine operation; therefore, wheatharvesting liberates immense numbers of fungalpathogen spores, many of which then can bedispersed long distances in wind currents.     

In-vitro phytochemical and pharmacological bio-efficacy studies on Azadirachta indica A. Juss and Melia azedarach Linn for anticancer activity

In this study, phyto-constituents, anti-bacterial and anticancer activity of Azadirachta indica A. Juss and Melia azedarach Linn was analyzed. High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) fingerprint profile of methanol extract of A. indica and M. azedarach was carried out. The present findings showed the presence of phytochemicals such as, steroids, alkaloids, phenols, flavonoids, saponins, tannins, anthraquinone and aminoacids in A. indica and M. azedarach extracts. HPLC profiling of methanolic extract of A. indica and M. azaderach revealed eleven and ten fractions of compounds were visualized in the form of peak. In TLC methanolic extract of A. indica was separated by eight distinct phenolic and three steroidal bands and M. azaderach showed sixteen distinct phenolic and three different steroidal bands. In antibacterial activity, Among the various extracts 50 µg/ml methanolic extracts of A. indica showed high activity against K. pneumoniae (14 mm) and M. azedarach showed high activity against S. aureus (15 mm). The results suggest that the crude methanolic extracts of A. indica and M. azedarach possess significant phytochemical properties compared to other extracts and hence the phytochemicals of M. azedarach and A. indica can be exploited for plant based anticancer and antimicrobial agents in the near future.     

Uromycladium tepperianum, the gall rust fungus from Falcataria moluccana in Malaysia and Indonesia

Batai (Falcataria moluccana) is a valuable tree species for forest plantations in Malaysia and Indonesia. Since 1993, a gall rust disease has caused severe damage to all growth stages, from seedlings in the nursery to mature trees in the field. To identify the fungus causing gall rust disease on F. moluccana in Malaysia and Indonesia, study of the mode of infection and changes in the anatomy of infected cells were carried out in the anatomy laboratory. The disease in Malaysia and Indonesia is caused by Uromycladium tepperianum. The fungus produces three longitudinally ridged teliospores on each head, with spores measuring 13–20 μm wide and 17–28 μm long. The fungus is microcyclic, completing its entire life cycle on F. moluccana. This study confirmed that the teliospores themselves cannot infect the host. Under favorable conditions, about 10 h after inoculation, teliospores germinate to produce basidiospores that form penetration pegs about 6 h later, and it is this peg which penetrates the host cells directly through the epidermis. Pycnia, recognized as small brown pustules, break through the epidermis about 7 days after inoculation.     

Marked enhancement in the artemisinin content and biomass productivity in Artemisia annua L. shoots co-cultivated with Piriformospora indica

A significant enhancement in artemisinin content, an important anti-malarial compound, has been achieved in Artemisia annua L. shoots by co-cultivating with Piriformospora indica, a mycorrhiza-like fungus. The in vitro shoots derived from nodal cultures of A. annua were implanted on four different culture media namely, (i) Murashige & Skoog (MS) basal, (ii) MS + 5 μM indole-3-butyric acid (IBA), (iii) MS + P. indica and, (iv) MS + 5 μM IBA + P. indica. After 2 months, it was observed that the cultures reared on MS + 5 μM IBA + P. indica showed optimum growth in terms of shoot and root proliferation over those cultured without P. indica. The average shoot number on MS + 5 μM IBA + P. indica was 17.83 ± 1.01 and on MS + P. indica alone was 12.75 ± 1.10. A drastic decline in shoot number was observed without P. indica which was 2.0 ± 0.12 on basal and 4.9 ± 1.52 on 5 μM IBA. Similarly, a maximum average of 16.83 ± 0.82 roots were achieved on MS + 5 μM IBA + P. indica which declined to 10.75 ± 1.02 on MS + P. indica. A further decrease in root number occurred in shoots without P. indica, their average being 2.5 ± 0.12 on basal and 8.91 ± 1.57 on 5 μM IBA. HPLC analysis of the aforesaid cultures revealed that the quantity of artemisinin was significantly higher (1.30 ± 0.03 %) in shoots cultured on 5 μM IBA + P. indica compared to those of control (0.80 ± 0.01 %).     

In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica

The present study was conducted for optimization of in vitro substrates under aseptic conditions for interaction of Piriformospora indica with the medicinal plant Coleus forskohlii. It aims to test the effects of different substrates on P. indica colonization as well as growth parameters of the in vitro raised C. forskohlii. Interaction of in vitro C. forskohlii with root endophyte P. indica under aseptic condition resulted in increase in growth parameters in fungus colonized plants. It was observed that P. indica promoted the plant’s growth in all irrespective of substrates used for co-culture study. The growth was found inferior in liquid compared to semisolid medium as well as there was problem of hyperhydricity in liquid medium. P. indica treated in vitro plantlets were better adapted for establishment under green house compared to the non treated plants due to fungal intervention.     

Potential geographical distribution of the red palm mite in South America

Among pests that have recently been introduced into the Americas, the red palm mite, Raoiella indica Hirst (Prostigmata: Tenuipalpidae), is the most invasive. This mite has spread rapidly to several Caribbean countries, United States of America, Mexico, Venezuela, Colombia and Brazil. The potential dispersion of R. indica to other regions of South America could seriously impact the cultivation of coconuts, bananas, exotic and native palms and tropical flowers such as the Heliconiaceae. To facilitate the development of efficacious R. indica management techniques such as the adoption of phytosanitary measures to prevent or delay the dispersion of this pest, the objective of this paper was to estimate the potential geographical distribution of R. indica in South America using a maximum entropy model. The R. indica occurrence data used in this model were obtained from extant literature, online databases and field sampling data. The model predicted potential suitable areas for R. indica in northern Colombia, central and northern Venezuela, Guyana, Suriname, east French Guiana and many parts of Brazil, including Roraima, the eastern Amazonas, northern Pará, Amapá and the coastal zones, from Pará to north of Rio de Janeiro. These results indicate the potential for significant R. indica related economic and social impacts in all of these countries, particularly in Brazil, because the suitable habitat regions overlap with agricultural areas for R. indica host plants such as coconuts and bananas.