Zoospores Research Articles

Distinct small RNAs are expressed at different stages of Phytophthora capsici and play important roles in development and pathogenesis.

Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici (P. capsici), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25-26nt in HY, SP, and ZO but distributed at 18-34nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici.

A Patched-Like Protein PsPTL Is Not Essential for the Growth and Response to Various Stresses in Phytophthora sojae.

Patched (Ptc) and Patched-related (Ptr) proteins containing sterol-sensing domains (SSD) and Patched domains are highly conserved in eukaryotes for lipid transport and metabolism. Four proteins containing predicted SSD and Patched domains were simultaneously found by searching the Phytophthora sojae genome database, and one of them was identified as a Patched-like (PTL) protein. Here, we investigated the biological function of PsPTL. The expression level of PsPTL was higher during mycelial and sporulation stages, compared to zoospore (ZO), cyst, and germinated-cyst stages, without significant change during infection. However, deletion of PsPTL using CRISPR/Cas9 had no significant effect on the growth, development, or virulence of P. sojae. Further investigations showed that PsPTL is not essential for P. sojae to cope with external stresses such as temperature, pH, oxidative and osmotic pressure. In addition, this gene did not appear to play an essential role in P. sojae’s response to exogenous sterols. The transcript levels of the other three proteins containing predicted SSD and Patched domains were also not significantly upregulated in PsPTL deletion transformants. Our studies demonstrated that PsPTL is not an essential protein for P. sojae under the tested conditions, and more in-depth research is required for revealing the potential functions of PsPTL under special conditions or in other signaling pathways.

Fosfito de potássio e suas implicações no controle de Phytophthora plurivora em faia

A faia (Fagus sylvatica) é uma das principais espécies das florestas na Europa e um dos principais entraves no cultivo dessa espécie é a ocorrência de doença causada por Phytophthora plurivora. Trabalhos apontam os fosfitos como boa alternativa para o controle de doenças causadas por oomicetos, entretanto, os mecanismos de ação ainda estão sendo estudados. O objetivo do trabalho foi avaliar o produto comercial à base de fosfito de potássio, Phytogard® no controle da doença causada por P. plurivora em faia, bem como avaliar os possíveis modos de ação do produto no patógeno. Foram utilizadas mudas de faia aspergidas com diferentes concentrações de Phytogard® e, posteriormente, inoculadas com o patógeno. Foram avaliados a incidência da doença, o consumo de água e a quantidade de DNA do patógeno nos tecidos das raízes do hospedeiro. Nos experimentos in vitro, foi determinado o crescimento micelial, produção de massa fresca de micélio e de zoósporos expostos ao Phytogard®. Avaliou-se também a perda de eletrólitos, peroxidação de lipídios e a atividade da enzima β-1,3-glucanase do micélio. A morfologia das hifas tratadas ou não com o Phytogard® foram observadas em microscópio eletrônico de varredura. O Phytogard®, em todas as concentrações aplicadas controla de maneira preventiva a doença causada por P. plurivora e também inibe o crescimento micelial e a produção de zoósporos. Além disso, modifica a morfologia das hifas, atua na permeabilidade de membrana e na síntese de parede celular do micélio do patógeno.

Transcription profiling and identification of infection-related genes in Phytophthora cactorum.

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.

First report of tobacco root rot caused by Phytophthora nicotianae in Mexico

During 2014 and 2015, symptoms of root rot were observed in tobacco fields located in Veracruz, Mexico. Phytophthora colonies developing rosaceous pattern were consistently isolated on potato dextrose agar (PDA) and V8 juice agar media. Sporangia were papillate, noncaducous, measuring 40 to 49 x 33 to 37 μm, and with short pedicels. The sporangial shape was ovoid or ellipsoid. Chlamydospores were mostly solitary, intercalary, or terminal, measuring 29-33 μm in diameter. The oomycete was identified as Phytophthora nicotianae based on descriptions of Gallegly and Hong (2008). To confirm the identification, genomic DNA from a representative isolate was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primers ITS5 and ITS4 (White et al., 1990). The resulting 896 bp sequence was deposited as GenBank Accession No. KR906059. BLASTn analysis showed a 97% similarity with those of P. nicotianae (GenBank Accession No. AB688370). Pathogenicity tests were performed twice on 20 healthy tobacco plants by immersion of roots in a zoospore suspension adjusted to 1 x 104 zoospores ml-1. Ten tobacco plants inoculated with sterile distilled water served as controls. Inoculated plants developed root rot symptoms after 10 days, whereas no symptoms were observed on the control plants. The oomycete present on inoculated plants was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of tobacco root rot caused by Phytophthora nicotianae in Mexico.

ALGAL LEAF SPOT OF LANSIUM PARASITICUM CAUSED BY CEPHALEUROS sp. IN THAILAND

Algal spot symptoms appeared on Lansium parasiticum (Osbeck) Sahni & Bennet leaves in late August, 2014 when rainy and warm weather favoured algal growth. The disease was found in 20% of surveyed L. parasiticum plants in Songkhla, Thailand, showing an orange to brown small, 1-4 mm circular scurf on the leaves. Thalli were sub-cuticular with sub- epidermal growth, and consequent necrosis of epidermal cells in the leaf tissue. Filamentous algal cells were short and cylindrical, 8-15×3-7 μm, with length/width ratio 1:1.1-4. Setae were short filaments with two to five cells, 100-180 × 3-7 μm. Sporangiophores, on the upper leaf surface, developed from the thalli of the algal colony. Sporangia were elliptical, 5-10×5- 8 μm. Gametangia were spherical to elliptical (8-15×3-7 μm), produced beneath the cuticle. Gametes were spheroidal (7-10×7-9 μm), zoospores were elliptical (5-10×5-8 μm). These morphometric traits were consistent with Cephaleuros sp. (Thompson and Wujek, 1997). The sequence of the 18S rDNA (GenBank accession No. LC086008), showed 94% identity to C. virescens (KM020142) and C. parasiticus (KM020146). In pathogenicity test, leaves of healthy L. parasiticum were wounded by rubbing with celite, inoculated by spraying with a suspension of algal thallus fragments (0.1 g/ml fresh thalli w/v) (Suto and Ohtani, 2011). Algal infection was evident after three months when small thalli (0.2 - 0.6 cm diameter) were observed. Reisolated algae were consistent with those originally isolated. To our knowledge, this the first report of algal leaf spot of L. parasiticum caused by Cephaleuros sp.

FIRST REPORT OF PHYTOPHTHORA NICOTIANAE ON CATHARANTHUS ROSEUS IN BANGLADESH

Annual vinca (Catharanthus roseus), also known as Madagascar periwinkle, is native to the island of Madagascar, and now common in many tropical and subtropical regions worldwide, including Bangladesh (Chevallier, 1996). It has long been cultivated for herbal medicine and as an ornamental plant. Symptoms of stem canker and leaf blight developed on C. roseus in a landscape planting at Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh, in June, 2014. To identify the pathogen, infected leaves were placed in water for 48 h, then the mycelia were transferred onto corn meal agar plates. Mycelial growth was patchy and irregular, coenocytic and tufted with an arachnoid branching habit, consistent with descriptions of Phytophthora nicotianae. The pathogen was also cultured on PARP medium (Ferguson and Jeffers, 1999) and V8 agar medium and pure cultures were established. Mycelial blocks (6 mm in diameter) were placed in sterile water for 3 days, then at 5 C for 15 min in the dark to stimulate production of sporangia and zoospores. Molecular identification was done by amplifying the ITS region of nuclear rDNA using ITS 4 and ITS 6 primer pairs. BLAST search revealed that the isolate had 100% identity with P. nicotianae (GenBank accession No. KT175508.1). The nucleotide sequence was deposited in GenBank under accession No. KU244700. Pathogenicity tests were conducted by inoculating Catharanthus leaves, previously wounded by pricking with a sterile needle, with 50 μl and 100 μl aliquots of zoospores. Foliar symptoms consistent with blight observed in the field developed on all the inoculated leaves after 72 h. Koch’s postulates were fulfilled by re-isolating the pathogen from the lesions on the inoculated leaves. To our knowledge, this is the first molecular identification of P. nicotianae on Catharanthus in Bangladesh.

Impact of soil salinity on fungal vector of rhizomania virus infecting beta vulgaris

Polymyxa betae Keskin is the only natural transmitting vector of the Beet necrotic yellow vein virus (BNYVV) among the cultivated sugar beet. This work aims to study the impact of salt stress on fungus-virus-host relationships. The fungal infected fine roots of sugar beet plant naturally infected with BNYVV were collected and treated with different salt concentrations (0, 2000, 4000, 6000 and 8000 ppm) of NaCl for one day prior to mixing it with a sterile soil. After virus symptoms appeared on leaves, disease severity has been determined, leave and roots tissues were collected for BNYVV detection. It was found that severe symptoms on sugar beet inoculated with treated roots by low salt concentrations (2000 and 4000 ppm) while at high salt concentrations, 8000 ppm less injury approximately as control treated with H2O. On the other hand it was found the cystosorial colonization was increased in low salt concentrations (2000 and 4000 ppm) while decrease in high salt concentrations (6000 and 8000 ppm) especially in 8000 ppm. The same trend results were observed in virus concentration in roots where as the BNYVV concentration was increased in low salt concentrations (2000 and 4000 ppm) while decrease in high salt concentrations (6000 and 8000 ppm). So the relation between capacity of fungal vector to infect the plant by virus and salinity concentration are antagonistic. Soil salinity extremes most often lead to decrease infection of BNYVV via effect on fungal zoospore. Key words: Polymyxa betae, sugar beet, rhizomania, beet necrotic yellow vein virus (BNYVV), salinity.

RNA-Seq reveals infection-related gene expression changes in Phytophthora capsici.

Phytophthora capsici is a soilborne plant pathogen capable of infecting a wide range of plants, including many solanaceous crops. However, genetic resistance and fungicides often fail to manage P. capsici due to limited knowledge on the molecular biology and basis of P. capsici pathogenicity. To begin to rectify this situation, Illumina RNA-Seq was used to perform massively parallel sequencing of three cDNA samples derived from P. capsici mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). Over 11 million reads were generated for each cDNA library analyzed. After read mapping to the gene models of P. capsici reference genome, 13,901, 14,633 and 14,695 putative genes were identified from the reads of the MY, ZO and GC libraries, respectively. Comparative analysis between two of samples showed major differences between the expressed gene content of MY, ZO and GC stages. A large number of genes associated with specific stages and pathogenicity were identified, including 98 predicted effector genes. The transcriptional levels of 19 effector genes during the developmental and host infection stages of P. capsici were validated by RT-PCR. Ectopic expression in Nicotiana benthamiana showed that P. capsici RXLR and Crinkler effectors can suppress host cell death triggered by diverse elicitors including P. capsici elicitin and NLP effectors. This study provides a first look at the transcriptome and effector arsenal of P. capsici during the important pre-infection stages.

Thematic Issue: Protists

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

Management of chili pepper root rot and wilt (caused by Phytophthora nicotianae) by grafting onto resistant rootstock

Root rot and plant wilting caused by Phytophthora nicotianae is a severe disease of chili pepper ( Capsicum annuum L.) in open fields and under greenhouse production in Tunisia. Chili pepper grafting for disease manage- ment is attracting increased interest in recent years. Using the tube grafting technique, different compatible scion/rootstock combinations were obtained with the wild-type pepper SCM334 and the local chili pepper cultivars ‘Beldi’ and ‘Baker’. SCM334 was resistant to P. nicotianae , while the cultivars Beldi and Baker were susceptible. Plant inoculations were performed with P. nicotianae zoospores, and severity of root rot was rated 30 days post- inoculation using a 0 (healthy plant) to 5 (dead plant) severity score. On SCM334 rootstock and with ‘Beldi’ or ‘Baker’ scions, the intensity of root rot was very low (mean score 0.1–0.2) and plants were healthy. However, with Baker or Beldi rootstocks and SCM334 scions, root rot was severe (mean score 3.1–4.6), leading to high numbers of wilting and dead plants. This severe root rot was similar to that observed on non-grafted plants of ‘Baker’ and ‘Beldi’ inoculated with the pathogen. Under greenhouse conditions, measurements of agronomic characters indicated non-consistent improvement of these features on the scion cultivar when SCM334 was the rootstock. Since plant foliage is not attacked by this pathogen, these results show that susceptible chili pepper scions grafted onto SCM334 rootstocks could be used for root rot management and improvement of pepper yields in P. nicotianae infested soils.

Bacteriolytic activity and growth of marine isolates of labyrinthulids on dead bacterial cells

Thirteen strains of labyrinthulids were isolated from the coastal area of Kagoshima Bay, Kagoshima Prefecture, Japan and from Batan Bay, Panay Island, the Phillippines by using diatom double-layer agar plates. The cells of labyrinthulid isolates grew axenically on the dead bacterial cells and extract plus egg yolk agar medium (NSBEY agar). Three representative isolates were demonstrated to belong to the labyrinthulid phylogenetic group (LPG) based on 18S rDNA sequence analysis. As bacteriolytic activity of labyrinthulid isolates was examined, it was found that they could lyze only the dead cells of gram-negative bacteria and not those of gram-positive bacteria during incubation in both agar and liquid media. The optimum temperature range for bacteriolysis was 25–31°C. Orange carotenoid pigment was accumulated during the stationary growth phase of strain 00-Bat-05, Philippine isolate, cultured in an L-shaped tube containing a bacterial dead cell suspension. Concomitant rapid cell movement of developing zoospores was observed.

고추의 역병 저항성 품종 개발을 위하여 Agrobacterium tumefaciens를 이용한 elicitin 유전자 도입

고추 형질전환은 Agrobacterium (LBA4404/pBI101 cyc600-syna-elicitin)을 이용한 cyc600 promoter에 구축된 elicitin 유전자의 형질전환시 shoot의 형성율은 수비초의 경우 3 mg/L zeatin과 0.05 mg/L NAA 함유 배지에서 11.1%, 4 mg/L zeatin과 0.05 mg/L NAA 함유 배지에서 12.8%였다. 전배양 3일, 공동배양 <TEX>$3{\sim}4$</TEX>일에서 재분화율이 높았다. 자엽으로부터 재분화된 형질전환체의 NPTII 유전자의 primer를 이용한 PCR 반응에서 형질전환된 재분화 식물체는 536 bp의 밴드를 확인하였다. Membrane에 blot하여 NPTII gene을 probe로 사용하여 Southern blot 분석에서 고추형질전환 식물체는 536 bp 부위에 강한 signal을 보였다. elicitin 유전자를 이용한 역병 저항성 형질전환체 수비초 <TEX>$T_{0}$</TEX>세대의 생육은 계통간에 다소 차이는 보였고, 계통 모두 생육은 저조한 반면 개화 및 수정 등의 임성은 정상적이었다. 자식을 통해 채종한 <TEX>$T_{1}$</TEX> 식물체의 유전자 도입을 확인하기 위하여 PCR을 수행한 결과 <TEX>$T_{0}$</TEX> 식물체 <TEX>$S1{\sim}S5$</TEX> 계통에서 elicitin 밴드가 나타나 형질전환체임을 확인하였고, <TEX>$T_{1}$</TEX>식물체인 S1-1 등 7계통에서 elicitin 밴드가 나타났고, S1-2 등 4계통에서는 밴드가 나타나지 않아 형질전환체가 후대에서 분리가 일어남을 확인할 수 있었다. Syn <TEX>${\alpha}$</TEX> clone을 이용하여 Southern blot analysis를 한 결과 band가 나타난 300 bp 정도의 위치에서 blot이 나오는 것을 관찰할 수 있었다. 위의 결과에서 cyc600 promoter-syn <TEX>${\alpha}$</TEX>의 construct가 수비초의 genomic DNA에 삽입되었는 것을 확인할 수 있었다. 고추형질전환체의 유묘에 역병균을 접종한 결과 유주포자 <TEX>$10^{3}$</TEX>개/mL에서 형질전환체의 저항성 계통선발이 가능하였다. The study was carried out to develop transformants resisting to Phyophthora blight disease in the domestic pepper cultivar Subicho. In transforming of syn600 promoter with elicitin gene using Agrobacterium (LBA4404/pBI101 syn600-syn<TEX>${\alpha}$</TEX>-elicitin) to cotyledons of pepper, rate of shoot formation in 'Subicho' was 11.1% in medium containing 3 mg/L zeatin and 0.05 mg/L NAA, and also 12.8% in medium containing combination of 4 mg/L zeatin and 0.05 mg/L MAA. For PCR reaction using elicitin gene primer of transformants regenerated from cotyledons, we detected a specific band of 536 bp, and also showed strong signal at position of 536 bp in accordance with NPTII gene used as probe in Southern blot. Transformants pepper shown resistance to blight fungus was inoculated to seedlings of the <TEX>$T_{1}\;and\;T_{2}$</TEX> transformants by concentration (density: zoo spore <TEX>$10^{3}/mL$</TEX>).

A Method to Monitor the Activity of "Phytophthora" spp. in the Root Zone of "Pistacia" spp.

A method was developed to monitor the formation of zoospores of Phytophthora species in the soil around potted host plants grown under greenhouse conditions. Inoculum of the pathogen was grown in vermiculite amended with hemp seed extract for 4 weeks at room temperature and 10 ml was placed around each plant. Pots were periodically flooded with water overnight with the drainage hole closed. Water was drained and collected next day and baited with citrus leaf discs for 48 h at room temperature and plated on a Phytophthora selective antibiotic medium. Baits colonized with Phytophthora were good indicators of the pathogen activity in the root zone of susceptible and resistant hosts.

Influences on survival of brown algal zoospore exerted by drifting particles

Influences on survival of brown algal zoospore exerted by drifting particles

Occurrence of parasexual cycle in Phytophthora parasitica following protoplast fusion.

Metalaxyl-resistant (M(superscript r)) and chloroneb-resistant (Cn(superscript r)) protoplasts of Phytophthora parasitica were fused, with heterokaryon formation rates ranging from 2.6×l0((superscript -3) to 8.2×l0(superscript -3). The heterokaryosis was confirmed in six of seven putative heterokaryons by the production of both M(superscript r) zoospores and Cn(superscript r) zoospores. and/or M(superscript r)+Cn(superscript r) zoospores. The 7th putative heterokaryon produced only Cn(superscript r) zoospores, probably because of the loss of the M(superscript r) chromosome during subculture. The heterokaryons produced normal-sized zoospores as well as large zoospores that were almost twice the size of normal-sized zoospores. Fifty percent of the normal-sized zoospores contained M(superscript r) and Cn(superscript r) genes. The results suggested an occurrence of diploidization after karyogamy of the heterokaryon and thus demonstrated the completion of events leading to a parasexual cycle following protoplast fusion.

TRANSCRIPT ACCUMULATION OF POLYGALACTURONASE INHIBITING PROTEIN (PGIP) FOLLOWING PATHOGEN INFECTIONS IN SOYBEAN

SUMMARY Proteins inhibiting fungal endo-polygalacturonase (PGIP) are constitutively expressed and localized on cell walls of most plant species. Induction of pgip transcripts following pathogen infection would demonstrate a role of PGIP in active plant defence mechanisms. We investigated pgip expression in hypocotyls of soybean seedlings (cvs ‘Sapporo’ and ‘Kure’) infected with the fungal pathogens Diaporthe phaseolorum var. caulivora, Sclerotinia sclerotiorum and an avirulent (race 1) and virulent (race 20) races of Phytophthora sojae. Two pgip transcripts were transiently expressed in all types of interaction with similar timing and maximum accumulation at 16-24 h after infection. However, soybean seedlings of both cvs infected with the virulent race 20 of P. sojae showed higher levels of pgip expression than seedlings infected with the avirulent race 1. A delay of pgip accumulation was observed when plants were inoculated with zoospores in place of mycelium of P. sojae. The presence of endo-polygalacturonase (endo-PG) in infected tissue was monitored both by immunological detection and by determining PG activity. PG was detected only in soybean seedlings infected with S. sclerotiorum and not in those infected with D. phaseolorum var. caulivora or P. sojae. The apparent inability in vivo of the virulent and avirulent races of P. sojae to produce PG suggests that PG-PGIP interaction is not required for the resistance response of soybean against this pathogen.

Establishment and Maintenance of Nuclear Position during Zoospore Formation inAllomyces macrogynus:Roles of the Cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis inAllomyces macrogynuswas assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS ofA. macrogynusis the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation.

Use of zoospores of Polymyxa betae in screening beet seedlings for resistance to beet necrotic yellow vein virus

A system to culture viruliferousPolymyxa betae and to produce zoospores is described. The zoo spores were used for inoculation of beet seedlings, grown in nutrient solution, in tests for resistance to beet necrotic yellow vein virus (BNYVV). On most occasions in a time course experiment, and with various zoospore cultures, the partially resistant cultivar Rima and the accession Holly-1–4 had virus concentrations similar to the susceptible cultivar Regina, but the virus concentration inBeta vulgaris ssp.maritima accession WB42 was significantly lower (P<0.05). ‘Regina’ could be distinguished from various resistant accessions by a significantly higher virus concentration (P<0.05) shortly after inoculation, or after transplanting the seedlings from the nutrient solution into sand. Results of screening for resistance to BNYVV, using zoospores for inoculation, did not correspond with results of a test in which infested soil was used.

Isozymal Relationships between Zoospores &amp; Vegetative Cells of Chlamydomonas Euryale

قورنت الأبواغ الحيوانية وخلالا التكاثر الخضري للكلاميدومونس أييوريال من خلال تفريدها كهربائيا على الجل من حيث بروتيناتها الذوابة الكلية ونظم التشابه الإنزيمي لإنزيمات الفاستيراز ، والفوسفاتاز القلوي ، مالات وهيدروجيناز ، جلوتامات ديهيدروجيناز ، ليوسين أمينوببتيداز ، بيرأ كسيداز ، تيروسيناز ، جلوتامات أكسالوأسيتات ترانس أميناز ، أميلاز ، لبيان مدى وجود اختلاف ، وعولجت النتائج طبقا لأقرب تطابق حسابي . دلت النتائج على وجود تطابق 100% لنوعي الخلايا في التشابه الإنزيمي للأميلاز فقط ، بينما بالنسبة للبروتينات الذوابة الكلية وأنزيمات الألفاستيراز، والمالات وديهيدروجيناز ، والجلوتامات وديهيدروجيناز ، والتيروسيناز والليوسين أمينوبيتيداز ، والبير كسيداز فكانت النتيجة بين 50-80% . بينما كانت النتيجة صفرا % بالنسبة للفوسفاتاز القلوي والجلوتامات أكسالو أسيتات ترانس أميناز وكان مدى التشابه المبني على البروتينات الكلية الذوابة وتسعة أنظمة للتشابه الإنزيمي 50%. وقد نوقشت الأسباب للنتائج المحتملة غير المتوقعة مع إبداء التوصيات .