Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region.

BACKGROUND: Analysis of phylogenetic relationship of 91 isolates of Phellinus noxius obtained from 46 plant species in Taiwan did not show distinct grouping based on ITS sequences. RESULTS: However, the ITS nucleotides showed 20 different kinds of variations including single nucleotide polymorphisms, deletion and insertion in ITS1 and ITS2, but none in 5.8 S. The Taiwanese isolates of P. noxius were dividable into long (type L), median (type M) and short (type S) groups based on ITS sequence length. Two isolates with identical ITS sequence belonged to types L. Type M with 72 isolates was further divided into 33 subtypes, while types S with 17 isolates was further divided into two subtypes. CONCLUSION: Phylogenetic analysis of ITS sequences among Phellinus species showed that isolates of P. noxius were in the same clade distinctly separated from other Phellinus species.

A new variant produced by Rhizoctonia solani AG1-IC isolate CH-1 with a new type of nuclei.

BACKGROUND: Isolate CH-1 of Rhizoctonia solani Kühn was commonly used in our studies of the pathogenicity and genetics of this pathogen. During the preparation of homokaryons through protoplast regeneration and tuft formation, a defective homokaryon was detected and a new variant was obtained. RESULTS: When tuft formation was used to identify the karyotic nature of single protoplast regenerants (SPRs) of Rhizoctonia solani AG1-IC isolate CH-1, one homokaryon type designated as A type and the parental heterokaryon designated as AB type were obtained. The homokaryon B type was not found. Various approaches were used to obtain SPRs, including from fast or slow growing protoplast regenerants, and from regenerants of protoplasts released from mycelia grown in different nutrient broths or at different temperatures. Without exception, all these SPRs were either homokaryon A or heterokaryon AB. Moreover, the SPRs obtained from different generations of SPRs, and from different generations of SPRs treated with lytic enzymes 3 to 4 times also were invariably either homokaryon A or heterokaryon AB. When single hyphal isolates were obtained from the tuft resulting from the pairing between homokaryon A and heterokaryon AB, only the heterokaryon and a variant were obtained. The variant did not form tuft when paired with parental heterokaryon AB or homokaryon A. Its protoplast regenerants gave rise to heterokaryon AB, homokaryon A and the variant, indicating that it is a new kind of heterokaryon. CONCLUSION: Inability to obtain homokaryon B despite numerous attempts suggests that the B type nuclei are probably defective and are dependent on A type nuclei for their multiplication. This is the first report of a heterokaryotic R. solani strain carrying a defective type of nuclei. A new variant which is a new kind of heterokaryon was obtained from the tuft resulting from the paring between the homokaryon A and the parental heterokaryon AB.

Characterization of a fungicidal substance produced by Eupenicillium brefeldianum isolated from soil for plant disease control and its significance in nature.

BACKGROUND: A fungus identified as Eupenicillium brefeldianum was isolated from soil amended with vegetable tissues. RESULT: When grown in liquid medium prepared from the same vegetable tissues, E. brefeldianum produced a substance capable of preventing disease development of leaf spots of mustard cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The inhibitory substance was fungicidal and was very stable under high temperature and extreme pH. It was soluble in polar solvents but not soluble in non-polar solvents, and did not have charges on its molecule. This is the first discovery of the production of a fungicidal substance by this fungus. CONCLUSION: Results from this study suggest the possession of a strong competitive saprophytic ability by E. brefeldianum, which in turn may explain the widespread occurrence of this fungus in soils.

Multiple resistance-activating substances produced by Humicola phialophoroides isolated from soil for control of Phytophthora blight of pepper.

BACKGROUND: Microorganisms capable of utilizing vegetable tissues for multiplication in soil were isolated, cultivated in liquid medium prepared from the same vegetable tissues, and tested for ability to activate resistance in pepper leaves against Phytophthora blight caused by Phytophthora capsici. RESULTS: Among the 121 microorganisms isolated, a fungus Humicola phialophoroides showed distinct ability to produce substances capable of activating resistance. The resistance-activating substances produced by H. phialophoroides were mostly retained in the mycelium, and were readily extracted from the mycelium powder with polar solvents. The extract was not inhibitory to zoospore germination or germ tube growth of P. capsici. In pepper leaves, the extract took only about 12 h to activate resistance against P. capsici. After activation, washing treated leaf surface with water did not have much effect on the resistance expression. In addition to being able to move from the upper leaf surface to lower leaf surface, the resistance-activating substances were capable of moving 5 mm acropetally and 10 mm basipetally in pepper leaves, Chromatography of the extract on silica gel column suggests that there are probably more than three components in the extract with resistance-activating ability. The resistance-activating activity of the mycelium extract was not affected by treatment with either cation or anion exchange resins, indicating that none of the active components have positive or negative charges on their molecules. CONCLUSION: Results show that H. phialophoroides is capable of producing multiple resistance-activating substances which are mostly retained in the mycelium. The study also indicates that none of the active components have positive or negative charges on their molecules.

Characterization of a fungistatic substance produced by Aspergillus flavus isolated from soil and its significance in nature.

A fungus capable of using vegetable tissues for multiplication in soil was isolated and identified as Aspergillus flavus based on morphological characteristics and sequence similarity of ITS and 28S. When grown in liquid medium prepared from the same vegetable tissues used in soil amendment, the isolate of A. flavus produced a substance capable of preventing disease development of black leaf spot of mustard cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The inhibitory substance was fungistatic, and was very stable under high temperature and high or low pH value. It was soluble in ethanol or methanol, moderately soluble in water, and insoluble in acetone, ethyl acetate or ether. The inhibitor is not a protein and has no charges on its molecule. This is the first discovery of the production of a fungistatic substance by this deleterious fungus. Results from this study suggest the possession of a strong competitive saprophytic ability by A. flavus, which in turn may explain the widespread occurrence of this fungus in soils. Production of a fungistatic substance when A. flavus was grown in medium prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soils.

Development of artificial conidia for ecological studies of Rhizoctonia solani in soil.

Artificial conidia of Rhizoctonia solani were developed by releasing protoplasts from young mycelia with lytic enzymes and by inducing cell wall formation in stabilizer solution. Conidia produced in this way were spherical with sizes ranging from 10 to 20µm in diameter. Artificial conidia were sensitive to soil fungistasis. Young hyphae originated from artificial conidia were also sensitive to fungistasis and mycolysis in soils. These results demonstrate that the previously reported insensitivity of R. solani to fungistasis and mycolysis in soils is due to special ability of propagules used rather than the inherited nature of the organism. Germination rates of artificial conidia on soils were inversely correlated with the amount of fungicide Flutolanil added. When germination of artificial conidia was used to detect suppressive soils, 3 out of 30 soil samples collected from different parts of Taiwan were suppressive to R. solani and all these suppressive soils were low in pH. Using artificial conidia for assay of fungicide activity in soil and detection of suppressive soils has the advantages of being fast and precise in comparison with relative hyphal growth. However, preparation of artificial conidia at this stage is tedious and time-consuming.

Activity and characterization of secondary metabolites produced by a new microorganism for control of plant diseases.

Microorganisms capable of utilizing vegetable tissues for growth in soils were isolated and their vegetable broth cultures were individually sprayed directly on leaves to test their ability to control Phytophthora blight of bell pepper caused by Phytophthora capsici. Liquid culture of Streptomyces strain TKA-5, a previously undescribed species obtained in this study, displayed several desirable disease control characteristics in nature, including high potency, long lasting and ability to control also black leaf spot of spoon cabbage caused by Alternaria brassicicolca. The extract was fungicidal to P. capsici but fungistatic to A. brassicicola. It was stable at high temperature and high pH. However, after exposure to pH 2 for 24h, the extract was no longer inhibitory to P. capsici although it was still strongly inhibitory to A. brassicicola. After treatment with cation or anion exchange resins, the extract lost its inhibitory effect against P. capsici but not A. brassicicola. The results suggest that the extract contained two different kinds of inhibitory metabolites, one against P. capsici with both positive and negative charges on its molecule and another against A. brassicicola with no charges on its molecule. The inhibitory metabolites were soluble in ethanol or methanol but not in water, ether or chloroform. They were dialyzable in the membrane tubing with molecular weight cut-off of 10,000, 1000 or 500 but not 100, indicating that the inhibitors have a molecular weight between 500 and 100. Results also showed that both inhibitors are not proteins.

Factors affecting protoplast formation by Rhizoctonia solani.

Novozym 234 was the most frequently used enzyme for production of Rhizoctonia solani protoplasts. Since manufacture of this enzyme was discontinued in the late 1990s, a new procedure was developed by testing lytic enzymes from Sigma and by examining factors affecting protoplast formation. The combination of 20 mg/mL Driselase and 10mg/mL lysing enzyme was effective in releasing protoplasts from R. solani. The optimal condition for enzyme treatment of mycelium was incubation at 37 degrees C for 15 min followed by 34 degrees C for 105 min. The amount of protoplasts produced was positively correlated with growth rate and negatively correlated with mycelial density. Under favorable conditions, R. solani mycelia released 1.68 x 10(6) protoplasts/mL that is comparable with that produced with Novozym 234. Among various media tested, the best solid medium for protoplast regeneration was 1% V-8 juice agar, while the best liquid medium was 10% potato dextrose broth.

Production of a fungistatic substance by Pseudallescheria boydii isolated from soil amended with vegetable tissues and its significance.

Four fungal isolates that were able to use vegetable tissues for multiplication in soil were isolated and identified as Pseudallescheria boydii based on morphological characteristics and ITS sequence similarity. When grown in broth prepared from the same vegetable tissues used in soil amendment, all these isolates of P. boydii produced a substance capable of reducing the disease incidence of black leaf spot of spoon cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The substance, which was fungistatic, was very stable under high temperature and high or low pH value. It was soluble in polar solvents and insoluble in non-polar solvents. Molecular weight estimation and ion exchange ability tests suggest that the fungistatic compound has a molecular weight between 500 and 1,000 and has no charge on its molecule. Results from this study suggest the possession of a strong competitive saprophytic ability by P. boydii, which in turn may explain the widespread occurrence of this human pathogen in soil. Production of a fungistatic substance when P. boydii was grown in broth prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soil.

Control of silverleaf whitefly, cotton aphid and kanzawa spider mite with oil and extracts from seeds of sugar apple.

Development of alternative methods for pest management is needed with the increased concern for adverse effects of pesticides for human health and the environment. The main goal of our study was to test the oil from seeds of sugar apple (Annona squamosa), an edible tropical fruit for pest control. The oil pressed out of seeds was as effective in controlling the silverleaf whitefly, Bemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae), infesting leaves of tomato plants in greenhouse conditions as the recommended insecticide, with the advantage of not being phytotoxic. When observed with a scanning electron microscope, the seed oil caused whitefly nymphs to shrink and detach from the leaf surface. Sugar apple seed oil was also very effective in controlling the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), on melon leaves and the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae), on soybean leaves. The study revealed the possibility of developing the oil from sugar apple seeds, an agricultural waste, into a broad spectrum product friendly to the environment and human health for crop pest management.

Control of silverleaf whitefly, cotton aphid and kanzawa spider mite with oil and extracts from seeds of sugar apple / Controle da mosca-branca, do pulgão do algodoeiro e do ácaro de kanzawa com óleo e extratos de sementes de fruta-do-conde

Development of alternative methods for pest management is needed with the increased concern for adverse effects of pesticides for human health and the environment. The main goal of our study was to test the oil from seeds of sugar apple (Annona squamosa), an edible tropical fruit for pest control. The oil pressed out of seeds was as effective in controlling the silverleaf whitefly, Bemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae), infesting leaves of tomato plants in greenhouse conditions as the recommended insecticide, with the advantage of not being phytotoxic. When observed with a scanning electron microscope, the seed oil caused whitefly nymphs to shrink and detach from the leaf surface. Sugar apple seed oil was also very effective in controlling the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), on melon leaves and the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae), on soybean leaves. The study revealed the possibility of developing the oil from sugar apple seeds, an agricultural waste, into a broad spectrum product friendly to the environment and human health for crop pest management.

É crescente a necessidade de desenvolvimento de métodos alternativos para o manejo de pragas com o aumento da consciência pública sobre os efeitos adversos de pesticidas à saúde humana e ao ambiente. O objetivo principal deste trabalho foi o de avaliar o óleo de sementes de fruta-do-conde (Annona squamosa), uma fruta tropical comestível, para o controle de pragas. O óleo prensado de sementes foi tão eficiente quanto o pesticida recomendado para controle da mosca-branca Bemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae) infestando folhas de tomate em casa-de-vegetação, com a vantagem de não apresentar fitotoxicidade. Em observações ao microscópio eletrônico de varredura, o óleo de semente induziu ao ressecamento das ninfas e o seu desprendimento da superfície da folha. O óleo de sementes de fruta-do-conde também foi eficaz no controle do pulgão do algodoeiro, Aphis gossypii Glover (Homoptera: Aphididae), infestando folhas de melão, e do ácaro de Kanzawa, Tetranychus kanzawai Kishida (Acari: Tetranychidae), em folhas de soja. Este estudo revelou a possibilidade de utilizar o óleo de sementes de fruta-do-conde, um sub-produto agrícola, como produto de largo espectro de ação mas seguro ao ambiente e à saúde humana, em programas de manejo de pragas agrícolas.

Occurrence of isolates of Phytophthora colocasiae in Taiwan with homothallic behavior and its significance.

Leaf blight and corm rot caused by Phytophthora colocasiae are the most devastating diseases of taro. Oospores of P. colocasiae have not been considered important in the survival in natural soil because the pathogen is heterothallic and there is essentially no chance for the presence of both A1 and A2 mating types in the same host tissue. During our recent survey of the mating type distribution of P. colocasiae in Taiwan seven homothallic isolates of Phytophthora were obtained from diseased taro leaves at Tsu Chi in central Taiwan. These organisms were identified as P. colocasiae based on morphological characteristics, ITS sequence homology and pathogenicity to taro plants. The homothallic isolates of P. colocasiae segregated into A1 and A2 types in addition to the original A1A2 type during asexual reproduction and vegetative growth. The homothallic isolate and the mixture of its A1 and A2 segregants produced abundant oospores in live tissue of taro petioles on or away from soil, indicating the possibility of oospores as a survival structure and the source of genetic variation in certain areas in nature.

Effect of Oriental Medicinal Plant Extracts on Spore Germination of Alternaria brassicicola and Nature of Inhibitory Substances from Speedweed.

Among 65 species of oriental medicinal plants tested, 39 contained substances inhibitory to conidial germination of Alternaria brassicicola, with the most inhibitory extract from speedweed (Polygonum perfoliatum). The inhibitory substances in dried speedweed were insoluble in water. However, the inhibitors were readily extracted with ethanol or methanol, but not with acetone, ether, or chloroform. The ethanol extract was very effective in controlling black leaf spot of spoon cabbage, Brassica campestris subsp. chinensis, caused by A. brassicicola. The inhibitory effect of the extract was not affected by treatment with anion exchange resins, but was partially reduced by cation exchange resins, indicating the presence of two inhibitory substances in the extract, one with a positive charge and the other with no charge on its molecule. The inhibitory substances in the extract were dialyzable with molecular weight minimums of 10,000 or 1,000, but not 500 or 100, suggesting that both inhibitors have molecular weights between 500 and 1,000.

Pythiogeton zizaniae, a new species causing basal stalk rot of water bamboo in Taiwan.

A new species, Pythiogeton zizaniae, was isolated from diseased water bamboo (Zizania latifolia) in central Taiwan. The organism formed a colony with scanty mycelia and mycelial aggregates on rye-water bamboo medium. Special treatments were required for production of sporangia which were terminal, noncaducous and mostly ovoid. Chlamydospores were absent. The fungus was homothallic. Oogonia produced on V-8 water bamboo medium in water were mostly globose to subglobose and each was attached with a club-shaped, monoclinous antheridium by the base of the oogonium stalk. Oospores were plerotic and globose to subglobose. Py. zizaniae caused death of water bamboo suckers but did not infect seedlings of corn, rice, wheat, sorghum, cucumber, tomato, soybean or water spinach. It also did not affect cucumber and tomato fruit, carrot roots or potato tubers.

Evidence for mitochondrial gene control of mating types in Phytophthora.

When protoplasts carrying metalaxyl-resistant (Mr) nuclei from the A1 isolate of Phytophthora parasitica were fused with protoplasts carrying chloroneb-resistant (Cnr) nuclei from the A2 isolate of the same species, fusion products carrying Mr nuclei were either the A2 or A1A2 type, while those carrying Cnr nuclei were the A1, A2, or A1A2 type. Fusion products carrying Mr and Cnr nuclei also behaved as the A1, A2, or A1A2 type. The result refutes the hypothesis that mating types in Phytophthora are controlled by nuclear genes. When nuclei from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species and vice versa, all of the nuclear hybrids expressed the mating type characteristics of the protoplast parent. The same was true when the nuclei from the A1 isolate of P. parasitica were fused with the protoplasts from the A0 isolate of Phytophthora capsici and vice versa. These results confirm the observation that mating type genes are not located in the nuclei and suggest the presence of mating type genes in the cytoplasms of the recipient protoplasts. When mitochondria from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species, the mating type of three out of five regenerated protoplasts was changed to the A1 type. The result demonstrated the decisive effect of mitochondrial donor sexuality on mating type characteristics of mitochondrial hybrids and suggested the presence of mating type genes in mitochondria. All of the mitochondrial hybrids resulting from the transfer of mitochondria from the A0 isolate of P. capsici into protoplasts from the A1 isolate of P. parasitica were all of the A0 type. The result supports the hypothesis of the presence of mating type genes in mitochondria in Phytophthora.

Pythium sukuiense, a new species from undisturbed natural forest in Taiwan.

A new species, Pythium sukuiense, was isolated from an undisturbed natural forest in northern Taiwan. The fungus produces sporangia indistinguishable from hyphae and very small oogonia and oospores. Oogonia were smooth and terminal or intercalary and attached with a single antheridium. Oospores were aplerotic, with an average size of only 11 µm.

Phytophthora cactorum can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.

Phytophthora cactorum grown on basal agarose medium or in liquid basal medium produced oospores after being transferred to water agarose. The numbers of oospores produced under such conditions depended on the age of the culture prior to exposure to nutrient deprivation. When the concentration of basal medium used for cultivation of P. cactorum was increased, the numbers of oospores produced after being transferred to water agarose was also increased. P. cactorum grown on basal agarose medium also produced oospores when its mycelial growth was restricted after reaching the edge of Petri plates. In 5 cm plates oospore formation occurred in the third week, whereas in 9 cm and 14 cm plates oospores appeared in the fourth week. Most oospores were formed near the edge of the plates. The non-saponifiables extracted from mycelia of P. cactorum grown in liquid basal medium were stimulatory to oospore formation by P. cactorum and Phytophthora parasitica, whereas the saponifiables were stimulatory to P. cactorum only. Extracts from culture filtrate and basal medium were not stimulatory to oospore formation by either fungus. When the non-saponifiables were fractionated by Florisil columm chromatography, only fraction 1 was not active. Fractions 2, 3 and 4 were stimulatory to oospore formation by both P. cactorum and P. parasitica. These results support the hypothesis that P. cactorum, and possibly other pythiaceous fungi as well, can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.

Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination.

Phytophthora cactorum did not form oospores on basal medium unless phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin) was added. After removal of putative sterols by aminopropyl column chromatography, the activities of lecithin and cephalin were increased 47- and 2.8-fold, respectively, thus confirming the previous reports that sterols are not essential for sexual reproduction in this organism. Thin-layer chromatography (TLC) of the commercial lecithin revealed the presence of an unknown inhibitory substance which, when added to the purified lecithin, caused a 50% reduction of oospore formation. Commercial cephalin also showed a twofold increase in activity after removal of putative sterols and the existence of an unknown inhibitor when it was subjected to TLC. Addition of the inhibitor to the purified cephalin completely inhibited the growth of the test organism. One sample of lecithin tested was not stimulatory to oospore formation. However, after washing with deionized water or NaCl solution, it induced the production of 17300 and 24450 oospores (100 µg)-1, respectively. The ability of cephalin to induce oospore formation was increased 2⋅3-fold by washing with deionized water and 8⋅3-fold by washing with NaCl solution. Like sterols, the digitonin precipitable component (digitonide) of the non-phospholipid fraction of commercial lecithin or cephalin was stimulatory to oospore formation of P. cactorum but not Phytophthora parasitica. However, the non-digitonide component was not only more active than the digitonide component, but also stimulatory to P. parasitica. Gas chromatography and mass spectrometry (GC-MS) analysis of the digitonide component from lecithin failed to detect any putative sterol contaminant. The amount of the putative sterol contaminant in the digitonide component from cephalin was also below the detection limit of GC-MS. When 0.01-10 ng cholesterol was added to basal medium discs each containing 100 fig cephalin, the numbers of oospores produced by P. cactorum and P. parasitica were not significantly changed. It is concluded that, in the fungi tested, sterols did not play any significant role in the stimulation of sexual reproduction by highly purified phospholipids.