Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Taxol,an anticancer drug produced by an endophytic fungus <Emphasis Type="Italic">Bartalinia robillardoides</Emphasis> Tassi,isolated from a medicinal plant, <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correa ex Roxb.

Taxol is an important anticancer drug widely used in the clinic. An endophytic fungus Bartalinia robillardoides (strain AMB-9) was isolated from Aegle marmelos, a medicinal plant and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed chromatographically and spectrometrically, for the presence of Taxol. The amount of taxol produced by this endophytic fungus was quantified by HPLC. It produced 187.6 μg/L of taxol which suggests that the fungus can serve as a potential material for genetic engineering to improve the production of Taxol. This fungal taxol isolated from the organic extract of this fungal culture, has strong cytotoxic activity towards BT 220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro, tested by Apoptotic assay.     

GAMETOPHYTE OF LYCOPODIUM DIGITATUM

Gametophytes of Lycopodium digitatum A. Braun are subterranean, mycorrhizal structures. Externally, they are conical and divided into three principal regions: tapering base, ring meristem, and gametangial cap. Internally, the endobiont is restricted to specialized tissues in the base. Gametangia typical of those described for L. complanatum are found along the cap margin near the ring meristem. Comparative analysis of these and other related gametophytes leads to the conclusion that Bruchmann's Type I (L. clavatum) and Type II (L. complanatum) gametophytes cannot be consistently separated on the basis of 1) mycorrhizal tissues, 2) form of gametophyte, and 3) number of neck canal cells in the archegonium, as he had proposed. Consequently, it is suggested on the basis of ring meristem and radial symmetry, mycorrhizal tissue specialization, and lack of sterile paraphyses or lobes that Type I and Type II gametophytes be merged as a single type. The now broadened clavatumcomplanatum type is restricted to subgenus Lycopodium (sensu Wilce) where it is the only known type.     

Gametophyte development and reproduction of <Emphasis Type="Italic">Argyrochosma nivea</Emphasis> (Pteridaceae)

The gametophyte of Argyroschosma nivea was studied, mainly focusing in its morphological development, and in the apogamous production of sporophytes. Some observations on the spores were also made. As far as it is known, this is the second species of the genus whose gametophytes are studied. The germination pattern followed the Vittaria type. The subsequent developmental processes followed the Ceratopteris type. Some of the gametophytes reached an adult stage with a cordate, symmetric shape, but most of them developed as irregular, lobed prothalli. The sporophyte emerged from the anterior part of the prothallus, without formation of gametangia. First, a cell became active and originated a proliferating area of small cells. From this area, long glandular hairs were formed followed by a projected conical cluster of cells. The cluster elongated into a sporophytic structure and its apex became progressively spatulate and finally trilobulate, with marginal, glandular hairs, stomata and tracheids continuously produced. This sporophyte secreted granules of white farina from its beginnings. The production of farina in the sporophyte but not in the gametophyte could help to support the idea of the segregation of this species from its traditional location in Notholaena to Argyrochosma, as farinose gametophytes seem to be a synapomorphy of the notholenoids, group that includes Notholaena but not Argyroschoma.     

GAMETOPHYTES AND SUBGENERIC CONCEPTS IN LYCOPODIUM

A critique of the Freeberg and Wetmore work on cultured Lycopodium gametophytes of L. selago, L. flabelliforme, and L. cernuum is presented. All three gametophytes are shown actually to be L. cernuum based on morphological and anatomical features of their sporophytes. A reassessment of characters in the genus demonstrates the taxonomic validity of the three groups proposed as subgenera within Lycopodium.     

The distribution of plasmodesmata and its relationship to morphogenesis in fern gametophytes

Fern (Onoclea sensibilis) gametophytes when grown in the dark form a linear file of cells (one-dimensional) called a protonema. In the light two-dimensional growth occurs which results in a heart-shaped prothallus one cell thick. The objective of this paper is to relate the most common pattern of cell division observed in developing gametophytes to the formation of the plasmodesmatal network. Since the prothalli are only two dimensional, we can easily determine from thin sections the total number and the density (number per unit surface area) of plasmodesmata at each developmental stage. As the prothallus grows the number of plasmodesmata increases 50-fold in the apical or meristematic cell. This number eventually reaches a plateau even though the density continues to increase with each new cell division. What is particularly striking is that both the number and density of plasmodesmata between adjacent cells is precisely determined. Furthermore, the pattern of plasmodesmata distribution is predictable so that (1) we can identify the apical meristematic cells by their plasmodesmata number, or density, as well as by their size, shape and location, (2) we can predict, again from plasmodesmata number, the location of a future wall of the apical cell prior to its actual formation, (3) we can show that the density of plasmodesmata in the triangular apical cell of the prothallus (14 plasmodesmata microns-2) is comparable to those reported for secretory glands which are known to have high rates of plasmodesmatal transport and (4) we can show that once the plasmodesmata have been formed during division, no subsequent change in the number of plasmodesmata occurs following cell plate formation.     

水蕨配子体发育的研究

研究了我国产水蕨(Ceratopteris thalictroides)配子体发育的全过程,水蕨孢子为四面体型,三裂缝,孢子萌发为向心型,丝状体4～8个细胞长,无明显的顶端细胞,丝状体顶部细胞同时分裂形成片状体,分生组织位于片状体一侧,使原叶体成为不对称的心脏形,原叶体发育为水蕨型,无毛状体,精子器和颈卵器较大,该配子体表现为较原始的性状.     

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.     

Comparative study on the gametophyte morphology and development of three paramo species of Jamesonia (Pteridaceae,Polypodiopsida)

The sexual phase of three species of Jamesonia (J. imbricata, J. rotundifolia and J. scammaniae) has been studied, from spore germination to gametangia formation, with special attention to the morphological development of prothalli. This is the first work to deal with gametophytes of species in this genus. All three species have trilete, tetrahedrical spores that germinate following the Vittaria type. The developmental pattern of J. imbricata and J. rotundifolia is intermediate between the Adiantum and Ceratopteris types, as the initial mitotic activity is assumed by an obconical apical cell, substituted later by a lateral meristem. In J. scammaniae, no apical cell or lateral meristem is formed, so the prothalli remain ameristic throughout the developmental process. Adult gametophytes are naked and variable in shape, in the same species ranging from typical cordate prothalli to irregularly lobed forms. Gametangia of normal shape and size was observed in both J. imbricata and J. rotundifolia, but not in J. scammaniae. Apogamy could be expected to occur in the genus, a phenomenon to be examined in the future. Comparisons are made with known species of related genera and results are discussed from an ecological perspective in the paramo environment.     

Isolation and identification of an anticancer drug,taxol from <Emphasis Type="Italic">Phyllosticta tabernaemontanae</Emphasis>, a leaf spot fungus of an angiosperm, <Emphasis Type="Italic">Wrightia tinctoria</Emphasis>

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (MID) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on MID medium (461 μg/L) followed by PDB medium (150 μg/L). The production rate was increased to 9.2 × 10³ fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.     

Growth and yield of groundnut (Arachis hypogaea L.) plants in soil inoculated with Aspergillus niger Van Tieghem

Aspergillus niger, a soil-borne fungus is a causative agent of hypocotyl malformations in infected groundnut (Arachis hypogaea L.) plants, but its effect on yield is unknown. This study sought to determine its effect on growth and yield. Seeds of Chinese and JL45 varieties were sown in soil inoculated with A. niger. Fresh and dry weights of the shoots and roots were taken at 10-day intervals. Nodule count was done at 30 days after emergence and subsequently at 10-day intervals. Pods of 20 plants each from inoculated and uninoculated soils were harvested. Growth was suppressed in plants grown on A. niger inoculated soil. Eight-day old plants grown in inoculated soil developed curvatures on their hypocotyls. Nodulation was suppressed (p < 0.05) in plants grown in inoculated soil. Although growth was suppressed in plants grown on inoculated soil, yield of both varieties of groundnut was not affected.     

Pseudomonas aeruginosa WS-1 for biological control of leaf blight disease of Withania somnifera

Withania somnifera (L.) Dunal, also known Indian ginseng is one of the most widespread tranquillizers tranquillisers used for the treatment of nervous disorders, intestinal infection, leprosy, and cancer; it also suffers a leaf blight disease caused by the fungus Alternaria dianthicola in various districts of South Bengal, India: Pseudomonas aeruginosa strain WS-1 isolated from the rhizosphere, showed both in vitro and in vivo antagonistic activity against the pathogen. The antifungal activity of the isolate has been found to be linked to theproduction of a siderophore, volatile substances (hydrocyanic acid), proteases and chitinases. Foliar application of a talc talc-based formulation of P. aeruginosa strain WS-1 to field grown W. somnifera reduced disease severity by 80% compared to non-treated control.     

Effect of Ploidy and Flowering Type of Red Clover Cultivars and of Isolate Origin on Severity of Clover Rot, Sclerotinia trifoliorum

Two complementary experiments were conducted in a controlled environment to elucidate the interactions between the fungus Sclerotinia trifoliorum Erikss. and red clover (Trifolium pratense L.). In one of these studies, two hardened diploid red clover cultivars (cvs) were inoculated with 20 isolates of S. trifoliorum of various geographic origins. In the other study, 20 red clover cvs, diploid or tetraploid, including late and medium‐late flowering types, were inoculated with two isolates of the fungus. Prior to inoculation, some plants were hardened by subjecting them to a low temperature and light treatment mimicking autumn conditions. Late flowering cvs were found more resistant than medium‐late ones. Isolates collected in the northern region, where late cvs are grown, were significantly more aggressive than isolates from southern locations, where medium‐late cvs are more prevalent. Such an adaptation has not previously been reported for this fungus. This is the first report concerning flowering type and resistance in red clover. Tetraploids were generally not more resistant than diploids. A hardening procedure for red clover plants was found to be a prerequisite for detecting the differences in disease resistance.     

Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures

This study describes the gel‐free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC‐MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 ( http://proteomecentral.proteomexchange.org/dataset/PXD000817 ).     

Endophytic fungus <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis> LF70 from <Emphasis Type="Italic">Huperzia serrata</Emphasis> produces Huperzine A

A strain LF70 endophytic fungus was isolated from the leaves of Huperzia serrata. The fungus was identified as Cladosporium cladosporioides LF70 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The strain could produce Huperzine A (HupA) identified through thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with authentic HupA. The amount of HupA produced by this endophytic fungus was quantified to be 56.84 μg/L by HPLC, which was higher than that of other reported endophytic fungi, Acremonium sp., Blastomyces sp., and Botrytis sp. Acetylcholinesterase inhibition activity of HupA produced by strain LF70 was also similar to authentic HupA in vitro. Isolation of such a fungus may provide a promising alternative approach to producing HupA, which is used in treating Alzheimer’s disease and preventing further memory degeneration.     

On Dark-Germination and Antheridium Formation in Anemia phyllitidis

Schraudolf's finding is confirmed that gibberellic acid induces antheridia in Anemia phyllitidis and Lygodium japonicum. The activity spectra of gibberellic acid and the native antheridiogen of Anemia phyllitidis are similar: Both induce antheridia in the tested species of the family Schizaeaceae but are inactive towards the tested representatives of other fern families. However, the native antheridiogens of two schizaeaceous species are more species-specific in their action than is gibberellic acid. Anemia medium cancels the light requirement for spore germination as is the case with gibberellic acid. Chromatographic studies indicate that the dark-germination-inducing factor is identical with, or very similar to, the earlier demonstrated antheridiogen. The specificities of the active factors towards dark-germination in A. phyllitidis and L. japonicum are similar to those encountered with antheridium formation. Anemia medium induces dark-germination and induces antheridia on the dark-grown protonemata to a concentration ca. 30 times lower than it induces antheridia on light-grown prothalli. The studies indicate that this differential activity results from a differential competence of the prothalli to react to the active substance in light and darkness.     

江南星蕨配子体形态发育的研究

用无机培养基和土壤培养基分别培养江南星蕨(Microsorium fortunei(Moore)Ching)孢子,显微镜下观察记录其孢子萌发及配子体形态发育过程.结果表明:孢子黄色,赤道面观豆形,极面观椭圆形,单裂缝,外壁具刺状纹饰.接种后7～12d孢子萌发,萌发类型为书带蕨型,配子体发育为槲蕨型.接种后25 d左右发育为片状体,片状体形成顶端细胞的时间较晚,有的甚至不形成.无机培养基培养的原叶体常在基部发生营养繁殖.毛状体出现在片状体形成之后,数量丰富,多为单细胞,分布于原叶体背腹面及边缘.接种后60 d左右发育形成幼原叶体,成熟原叶体呈心脏形.接种后80 d左右开始有性器官出现,精子器的出现较颈卵器早10d左右.颈卵器成熟后,颈部常向原叶体基部倾斜或弯曲.     

Caloplaca erodens [sect. Pyrenodesmia], a new lichen species from Italy with an unusual thallus type

Caloplaca erodens is a new species of sect. Pyrenodesmia, characterised by an orbicular, sorediate, bluish-grey thallus which is endolithic but emerges at the periphery with a white, K−, obscurely lobate prothallus. The species is frequent on calcareous outcrops and walls of isolated churches and ruins of the Central Apennines (Sibillini, Gran Sasso), where it may occur in large monospecific populations, from 1000 to 2500 m asl., and is also known from dry sites of the southern Alps. It has been found with apothecia only in the type locality, being predominantly sterile. The reproduction of this lichen is evidently linked to the release of fragments of clusters of photobiont cells and mycobiont hyphae which are continuously exposed with the dissolution of the substratum. Readily distinguished from the apparently similar endolithic C. alociza (which is characterised by numerous apothecia, black, K+ purple prothallus, and esorediate thallus), C. erodens probably belongs to the C. circumalbata complex, whose taxa are always epilithic but have a white, K− prothallus. The phylogenetic position of the new species within sect. Pyrenodesmia as inferred by ITS sequences of the nuclear ribosomal DNA is shortly discussed.     

Johnkarlingia,a new genus of the synchytriaceae

A chytridiaceous fungous species was found parasitic in the roots of cauliflower (Brassica oleracea L. var. botrytis) and cabbage (Brassica oleracea L. var. capitata) in the heavy soil fields of Varanasi, U.P. The morphology of resting sporangia and life cycle of the pathogen revealed that no fungus species has hitherto been described befitting its developmental pattern. A new genus Johnkarlingia Pavgi & Singh has been proposed to accommodate the fungus with Johnkarlingia brassicae Singh & Pavgi as its type species. The taxonomy and affinities of the genus are discussed.     

Studies on the Relationship of Cowpea mosaic virus and its Vector Aphis cracdvora Koch

Chillies or Green peppers (Capsicum annuumL.) are extensively grown in this country during rainy and winter seasons. The crop brings good profit for the grower as it is widely used in green as well as in dried condition. A fruit spot disease of this valuable vegetable was noticed during the cold months of 1962 at Phule Market, Poona, on the sweet variety (which is usually bigger in size), stored in bamboo baskets and crates. On microscopical examination of such spots the fungus appeared to be a species of Helminthosporium. The pungent varieties of chilies such as‘Gauran’(long in size) and‘Dharwar round’wert; found to be free from infection. Affected fruits incubated in moist chambers gave abundant sporulation of the causal organism. Ripened and red chili-fruits were free from such infection in the markets. The present paper gives an account of the etiology of the disease, pathogen city and cultural behaviour of the fungus.