Mixed Spore Research Articles

Establishment of an Efficient Screening Methods for Resistance of Chinese Cabbage to Clubroot Disease

Clubroot caused by <i>Plasmodiophora brassicae</i> is an important disease of crucifer crops worldwide. This study aimed to establish an efficient screening method to determine resistant cultivars of Chinese cabbage against <i>P. brassicae</i>. To do this, we investigated the virulence of seven <i>P. brassicae</i> isolates using seedlings of susceptible Chinese cabbage cultivar. The isolates exhibited different virulence in the plants and were divided into three groups based on their virulence. When we explored the disease occurrence in Chinese cabbage seedlings according to photoperiod after inoculation of <i>P. brassicae</i> and incubation temperature, the plants with all-day light showed higher disease severity than seedlings cultivated under 14 hr of light a day. The occurrence of clubroot disease was most severe at 25<sup>o</sup>C, followed by 20<sup>o</sup>C and 18<sup>o</sup>C, but the fresh weight of clubroot of the seedlings cultivated at 20<sup>o</sup>C was the highest, followed by plants grown at 25<sup>o</sup>C and 18<sup>o</sup>C. When the seedlings of two commercial resistant cultivars were inoculated with the mixed spore suspensions of two different pathotype isolates of <i>P. brassicae</i>, disease severity increased as the spore concentration of the susceptible <i>P. brassicae</i> isolate among the two strains increased, suggesting that the clubroot development by different pathotype isolates was independent and not influenced by each other. Taken together, our results provide a faster and more accurate screening methods to determine the resistance of Chinese cabbage against <i>P. brassicae</i>.

Automatic Detection of Rice Blast Fungus Spores by Deep Learning-Based Object Detection: Models, Benchmarks and Quantitative Analysis

The severity of rice blast and its impacts on rice yield are closely related to the inoculum quantity of Magnaporthe oryzae, and automatic detection of the pathogen spores in microscopic images can provide a rapid and effective way to quantify pathogen inoculum. Traditional spore detection methods mostly rely on manual feature extraction and shallow machine learning models, and are mostly designed for the indoor counting of a single spore class, which cannot handle the interference of impurity particles in the field. This study achieved automatic detection of rice blast fungus spores in the mixture with other fungal spores and rice pollens commonly encountered under field conditions by using deep learning based object detection techniques. First, 8959 microscopic images of a single spore class and 1450 microscopic images of mixed spore classes, including the rice blast fungus spores and four common impurity particles, were collected and labelled to form the benchmark dataset. Then, Faster R-CNN, Cascade R-CNN and YOLOv3 were used as the main detection frameworks, and multiple convolutional neural networks were used as the backbone networks in training of nine object detection algorithms. The results showed that the detection performance of YOLOv3_DarkNet53 is superior to the other eight algorithms, and achieved 98.0% mean average precision (intersection over union > 0.5) and an average speed of 36.4 frames per second. This study demonstrated the enormous application potential of deep object detection algorithms in automatic detection and quantification of rice blast fungus spores.

Heat-stable spores of carotenoid-producing Bacillus marisflavi and non-pigmented Bacillus subtilis cooperatively promote growth, quality, and gut microbiota of white-leg shrimp.

We evaluated the benefits of heat-stable carotenoid-producing Bacillus marisflavi SH8 spores individually and in combination with non-pigmented Bacillus subtilis SH23 spores on growth, colour change, nutritional content, innate immunity, and gut microbiota of white-leg shrimp. White-leg shrimp (Litopenaeus vannamei; n= 30 per tank; 2 tanks per group) were provided feed without (control group) or with SH8, SH23, or mixed spores (total, 1× 106 cfu/g pellet) for 28 d. The SH8 and SH8-23 combination groups had significantly higher specific growth rates (9.6 and 11.0%), improved red-colour score (4 scores), astaxanthin concentration (1.8- and 2.3-fold), lipid contents (30 and 50%), and superoxidase dismutase activity (8.5 and 12.3%) than that of the control group. Analysis of shrimp's gut microbiome using 16S rRNA metagenome sequencing revealed increased abundance of four useful species and reduced abundance of four harmful species in the combination group than in the control group. Heat-stable Bacillus spore combination improved growth parameters, nutrient content, red-colour score, live counts, and abundance of useful bacteria in the gut of L. vannamei. This is the first study to show the benefits of combining highly heat-stable pigmented and non-pigmented Bacillus spores and their possible mechanisms in a shrimp model.

Detection of wheat scab fungus spores utilizing the Yolov5-ECA-ASFF network structure

Rapid detection and identification of Fusarium germinate spores play a vital role in the early prediction and effective management of wheat scab disease. This study proposed an improved Yolov5-ECA-ASFF target detection algorithm that addressed the challenges of small size and precise localization of spore image targets. The algorithm incorporated the attention mechanism module (ECA-Net) and adaptive feature fusion mechanism (ASFF) into the feature pyramid structure of YOLO, effectively tackling issues related to small size, limited characteristics, and unclear attributes of F. germinate spores. The results demonstrate that the proposed model achieved an average recognition accuracy of 98.57% for F. graminearum spores, surpassing the original Yolov5s algorithm’s mAP value by 6.8%. The proposed method outperformed other mainstream target detection networks like Yolov4 and Faster-RCNN. It also exhibited excellent recognition outcomes in scenarios involving multiple targets and complex backgrounds, while maintaining model robustness even when faced with similar appearance, morphology, and color characteristics of various scab spores. In conclusion, this method accurately detected and identified wheat scab spores in the presence of a variety of mixed spores, providing crucial technical support for automated detection of wheat scab spores and early prediction of wheat scab outbreaks under complex field environments.

Viral cross-class transmission results in disease of a phytopathogenic fungus

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Spore inactivation on solid surfaces by vaporized hydrogen peroxide-Influence of carrier material surface properties.

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillussubtilis and Bacillusatrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role.

Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi.

To clarify the relationship between neutral lipid content and cordycepin accumulation in Cordyceps militaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis.

Arbuscular mycorrhizal fungi protect native woody species from novel weapons

Arbuscular mycorrhizal fungi (AMF) play important roles in plant community structure and ecosystem functioning. The allelopathic disruption of arbuscular mycorrhizal mutualism is a potentially powerful mechanism by which non-native species can negatively impact native plant diversity. However, there is limited understanding of this mechanism on woody species in forest ecosystems. In this study, we carried out a set of experiments to explore the allelopathy of Eucalyptus urophylla on mycorrhizal mutualists. First, we examined allelopathic effects on the mycorrhizal growth responses of woody species by collecting leachates from the understory of an E. urophylla plantation. Second, we examined if AMF could counteract the allelopathy of E. urophylla by treating the target species with and without aqueous extract of E. urophylla and AMF inoculum. We conducted a third in vitro experiment to characterize the effects of identified putative allelochemicals (IPAs) of E. urophylla on AMF spore germination, namely Glomus mosseae, Claroideoglomus etunicatum and mixed spores extracted from field soil. There was a positive correlation between the stimulatory effects of natural leachates of E. urophylla and the mycorrhizal growth responses of target woody species. AMF could counteract the negative impact of E. urophylla allelopathy at a relatively low concentration. The IPAs of E. urophylla had variable effects on germination of AMF spores, from stimulatory to inhibitory, depending on chemical types and AMF species. Moreover, the AMF G. mosseae was the least sensitive to allelopathic inhibition of E. urophylla IPAs. This study does not support the hypothesis that allelopathy degrades mycorrhizal symbioses. Our study first provides preliminary evidence for a positive correlation between allelopathy and mycorrhizal growth responses and suggests that higher mycorrhizal growth responses could better protect woody species from allelopathic inhibition in E. urophylla plantations.

Promoting water deficit tolerance and anthocyanin fortification in pigmented rice cultivar (Oryza sativa L. subsp. indica) using arbuscular mycorrhizal fungi inoculation.

Drought or water deficit is a major abiotic stress that can reduce growth and productivity in the rice crop especially in the rain-fed areas, which face long-term water shortage. The objective of this investigation was to promote the drought tolerant abilities in pigmented rice cv. 'Hom Nil' at booting stage using arbuscular mycorrhizal fungi (AMF)-inoculation, mixed spores of Glomus geosporum, G. etunicatum and G. mosseae in the soil before rice seedling transplantation. At booting stage, the AMF-inoculated (+AMF) and AMF-uninoculated plants (-AMF) were subjected to control (well-watering; 46.6% SWC) and water deficit condition (14 days water withholding; 13.8% SWC). Colonization percentage in the AMF-inoculated root tissues were evidently proved in both with and without water deficit conditions, leading to elevate total phosphorus in root and leaf tissues. Interestingly, sucrose and total soluble sugar concentration in the flag leaf were increased by 5.0 folds and 1.5 folds, respectively in the plants under water deficit (WD). Free proline was accumulated in flag leaf when exposure to water deficit, subsequently regulated by AMF-inoculation. Total soluble sugar and free proline enrichment in 'Hom Nil' was a major mode of osmotic adjustment to control osmotic potential in the cellular level when exposed to water deficit, leading to maintained photosynthetic abilities and growth performances. Concentration of chlorophyll b in AMF-inoculated plants under water deficit stress was retained, causing to improve chlorophyll fluorescence and net photosynthetic rate. Shoot height and number of tillers were significantly declined by 12.5% and 11.6%, respectively, when subjected to WD. At the harvest, grain yield, panicle dry weight and fertility percentage of AMF-inoculated rice from WD were greater than those without AMF by 1.5, 3.9 and 2.4 folds, respectively. Cyanidin-3-glucoside and peonidin-3-glucoside concentrations in pericarp were enriched in the grain derived from AMF-inoculation with water deficit stress. Overall growth characters and physiological adaptations in 'Hom Nil' grown under water deficit condition were retained by AMF inoculation, resulting in enhanced yield attributes and anthocyanin fortification in rice grain.

Experimental Investigations on Biological Resistance of Surface Modified Coir Geotextiles

Results of detailed experimental studies carried out to investigate the resistance of coir geotextiles modified using cashew nut shell liquid (CNSL) to attack of biological organisms are reported. A mixed spore suspension composed of four fungal stains, Chaetomium indicum, Curvularia lunata, Aspergillus fumigatus and Penicillium rubrum was used to study microbial susceptibility of coir geotextiles in humid warm atmosphere. Soil burial studies were carried out under controlled conditions for a period of 240 days to learn the behaviour of geotextiles in contact with specially prepared soil containing a variety of microorganisms. A field study was carried out to investigate the behaviour of coir geotextiles in subterranean termite mounds. The improved properties of modified coir geotextiles were substantiated on the basis of weight loss, moisture absorption, tensile strength and surface morphology. The results show that biological resistance of coir geotextiles was greatly improved by modification with CNSL. Tensile strength of unmodified samples reduced to 19 % whereas modified geotextiles retained 76 % of the initial tensile strength at the end of 240 days of soil burial. SEM images affirm that modification of coir with CNSL obtained in the present work could close the pores on fibre surface and delay biological degradation considerably.

Effect of Water Activity, Temperature, and Mixed Fungal Spore Interactions on Ochratoxin A Production by Aspergillus carbonarius

The purpose of this work was to investigate the potential of two nontoxigenic Aspergillus section Nigri species (Aspergillus tubingensis and Aspergillus japonicus) to influence the in vitro ochratoxin A (OTA) production of three toxigenic Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33) from Greek vineyards of different geographical areas. OTA accumulation was evaluated by inoculation of 0:100, 25:75, 50:50, 75:25, and 100:0 ratios of mixed spore suspensions on a synthetic grape juice medium for up to 28 days at different temperatures (15, 20, and 25°C), water activity (aw) levels (0.95 and 0.98 aw) and incubation time (7, 14, 21, and 28 days). Results confirmed that environmental factors and fungal species had a significant effect on OTA production. Specifically, maximum OTA concentration for Ac-28 (3.21 μg g−1) and Ac-29 (7.69 μg g−1) was observed at 20°C/0.98 aw and for Ac-33 (9.13 μg g−1) at 15°C/0.95 aw, regardless of incubation time. Moreover, A. tubingensis had no significant influence on OTA concentration of all toxigenic isolates assayed, regardless of temperature, aw, and incubation time. On the other hand, the presence of A. japonicus slightly inhibited OTA production of Ac-29 and Ac-33, while for Ac-28, stimulation of OTA was observed in some cases. Overall, lower aw levels reduced OTA accumulation for Ac-28 and Ac-29, regardless of temperature, inoculum ratio, and time. On the contrary, for Ac-33, low aw increased OTA production, regardless of the investigated parameters. The importance of this study concerns the understanding of interspecific interactions on OTA diffusion by A. carbonarius in an attempt to find ways to prevent the presence of toxins in grapes and their derivatives.

뿌리혹병 및 시들음병에 대한 저항성 양배추와 브로콜리 유전자원 탐색

Brassica olerace의 주요 병해인 뿌리혹병과 시들음병에 대한 저항성 유전자원을 발굴하기 위하여, 농업유전자원정보센터로부터 제공받은 양배추(B. oleracea var capitata) 유전자원 60개와 브로콜리(B. oleracea var italica) 유전자원 6개의 뿌리혹병과 시들음병에 대한 저항성을 검정하였다. 유전자원의 뿌리혹병에 대한 저항성 검정을 위해서, 분양 받은 유전자원들의 유묘에 뿌리혹병균 Plasmodiophora brassicae의 포자현탁액을 접종하였다. 실험한 유전자원 중 양배추 유전자원 4개는 중도저항성을 보였으며, 이들 중 양배추 유전자원 'K166220'는 가장 높은 저항성을 나타냈다. 나머지 유전자원들은 감수성을 보였다. 한편, 유전자원의 시들음병에 대한 저항성을 검정하기 위하여 분양 받은 유전자원 유묘의 뿌리를 Fusarium oxysporum f. sp. conglutinans 포자 현탁액에 침지하여 접종하였다. 실험한 유전자원 중 양배추 유전자원 17개와 브로콜리 유전자원 5개는 저항성, 양배추 유전자원 16개는 중도저항성 그리고 나머지 유전자원은 감수성이었다. 특히 양배추 유전자원 3종('IT227115', 'K161791', 'K173350')과 브로콜리 유전자원 2종('IT227100', 'IT227099')은 시들음병에 대하여 높은 저항성을 보였다. 본 연구에서 확인된 뿌리혹병과 시들음병 저항성 유전자원들은 뿌리혹병과 시들음병 저항성 육종에 이용될 수 있을 것이다. Clubroot and Fusarium wilt of cole crops (Brassica oleracea L.) are destructive diseases which for many years has brought a decline in quality and large losses in yields all over the world. The breeding of resistant cultivars is an effective approach to reduce the use of chemical fungicides and minimize crop losses. This study was conducted to evaluate the resistance of 60 cabbage (B. oleracea var. capitata) and 6 broccoli (B. oleracea var. italica) lines provided by The RDA-Genebank Information Center to clubroot and Fusarium wilt. To investigate resistance to clubroot, seedlings of the genetic resources were inoculated with Plasmodiophora brassicae by drenching the roots with a mixed spore suspension (1 : 1) of two isolates. Of the tested genetic resources, four cabbage lines were moderately resistant and 'K166220' represented the highest resistance to P. brassicae. The others were susceptible to clubroot. On the other hand, to select resistant plants to Fusarium wilt, the genetic resources were inoculated with Fusarium oxysporum f. sp. conglutinans by dipping the roots in spore suspension of the fungus. Among them, 17 cabbage and 5 broccoli lines were resistant, 16 cabbage lines were moderately resistant, and the others were susceptible to Fusarium wilt. Especially, three cabbage ('IT227115', 'K161791', 'K173350') and two broccoli ('IT227100', 'IT227099') lines were highly resistant to the fungus. We suggest that the resistant genetic resources can be used as a basic material for resistant B. oleracea breeding system against clubroot and Fusarium wilt.

Low cost medium for spore production of Bacillus KKU02 and KKU03 and the effects of the produced spores on growth of giant freshwater prawn (Macrobrachium rosenbergii de Man).

In order to extend the shelf life of 2 high potential Bacillus probiotic isolates which were Bacillus KKU02 and Bacillus KKU03, the spore forms of these 2 Bacillus isolates were studied for using as probiotic instead. The low cost medium for spore production of these 2 Bacillus isolates was examined in order to produce probiotic spores for feeding the shrimps. It was found that cassava at 100 g L(-1) and supplemented with 20.0 g L(-1) dextrose, 0.1 g L(-1) MgSO4 and 2.0 g L(-1) (NH4)2SO4 showed the highest spore concentration at about 1 x 10(8) CFU mL(-1). The effects of feeding these 2 Bacillus spores on the growth of giant freshwater prawns were further examined. The spores of Bacillus KKU02 and Bacillus KKU03 (-10(7) spore mL(-1)) in pure and mixed culture forms were mixed with commercial prawn feed (200 mL kg(-1)) to give six feed treatments. Body length and weight of the prawns in mixed spore culture tanks after rearing for 90 days (13.5 cm and 59.8 g, respectively) were significantly higher (p = 0.05) than other treatments. The treated prawns were further challenged with Aeromonas hydrophila for 7 days. The percentages of survival after the challenge in the prawns fed with the mixed spores (46.8%) were also found significantly higher (p = 0.05) than others groups, except the mixed live cell treatment (60%). These results indicated that the spores of Bacillus KKU02 and Bacillus KKU03 had a high potential for using as commercial probiotics.

Mutation Screening of Production Strains of Spinosad Based on Ribosome Engineering Theory through ARTP

Based on the ribosome engineering theory,the atmospheric and room temperature plasma( ARTP) was used in screening of Saccharopolyspora spinosa. Taking the strain Saccharopolyspora spinosa QYLZ 88912 as the original strain,choose the spore suspension of three different irradiation time after ARTP mutation, then mix them to increase the resistance screening probability. A high-yield strain S. spinosa Sg200Rif110St40Er90-028 was obtained after screening to the mixed spore suspension. This strain had the resistance of sulfaguanidine, rifampin,streptomycin,erythromycin. Its production of spinosad reached 1516. 93 mg / L under the level of shake flask fermentation through 7 days,which was 148. 37% higher than that of the original strain S. spinosa QYLZ 88912. The subculture experiments indicated that the hereditary characteristic of high productivity of S. spinosa Sg200Rif110St40Er90-028 was relatively stable. The fermentation materials of spinosad producing strain were optimized by the response surface methodology based on the single factor experiment. Under the optimized medium,the spinosad was increased by 286. 71% compared with the previous condition.

Effect of environmental factors on in vitro and in situ interactions between atoxigenic and toxigenic A. flavus strains and control of aflatoxin contamination of maize

The potential of three atoxigenic strains from different geographical origins in Africa were examined for in vitro and in situ competitiveness against two toxigenic strains of Aspergillus flavus and subsequent inhibition of aflatoxin B1 (AFB1) production under different environmental conditions. Temperature, water activity (a w) and substrate influenced the types of interaction between the three AFL− and two AFL+ strains. The competitiveness and AFB1 reduction ability of the three atoxigenic strains when interacting with the two toxigenic strains were evaluated by inoculation of 100, 25:75, 50:50, 75:25 and 100% ratios of mixed spore suspensions in vitro on malt extract and milled maize agars over 28 days and in situ on stored maize grain for 14 days, respectively at 0.99, 0.96 and 0.90 a w. For all the treatments, the effect of a w and inoculum ratio and their interaction was highly significant. Toxin inhibition was >80% in vitro at both 0.99 and 0.96 a w. In situ AFB1 reduction was influenced by the toxigenic strain assayed, a w and the inoculum ratio. Where control was achieved, it was more variable at 0.96 a w, while with more stringent water stress conditions (0.90 a w) the percentage inhibition was up to 77.2%. The study shows the importance of including environmental factors in screening and identifying effective atoxigenic strains for control of AFs (aflatoxins).

A report on pharmacognostical evaluation of four Adiantum species, Pteridophyta, for their authentication and quality control

Genus Adiantum L., Pteridaceae, is an important fern used in traditional systems of medicine. Different species of Adiantum are known as avenca in Brazil; hansraj/hanmspadi in India; maiden hair fern in English. The present study aims to develop the quality control parameters of four similar looking Adiantum species viz. A. capillus-veneris L., A. lunulatum Burm. f., A. peruvianum Klotzsch, and A. venustum D. Don. Standard methods for macro-microscopic evaluation, physicochemical parameters and HPTLC were used for authentication and identification. The salient distinctive characters under the microscope are the presence of slightly wavy elongated epidermal cells in A. capillus-veneris; epidermal cells strongly wavy in A. lunulatum; star shaped epidermal cells and mixed spores of regular and irregular shaped in A. peruvianum; stomata on both the surfaces of pinnule, absence of spinulus spores only in A. venustum. In addition, rachis anatomy showed different cellular and stellar characteristics as identifying characters of aforesaid four Adiantum species. Physico-chemical parameters and HPTLC finger print profiles along with stigmasterol and lupeol play significant role for the quality evaluation of raw drugs. The above finding will serve the purpose of quality control and assurance for the future studies.

High Frequency Sporophytes Regeneration from the Spore Culture of the Endangered Aquatic Fern <i>Isoetes coreana</i>

Using a mixed culture of megaspores and microspores from I. coreana, we established high frequency sporophyte regeneration system. After 20 days of culturing in MS basal medium, microscopic examination showed significant morphological changes and the microspore released numerous small vesicles into the culture medium. Megaspores also showed dramatic morphological changes during its incubation time in culture. The spore wall was cracked by the expansion of the megaspore (about 2 times increase in diameter). Simultaneously, brown spots were observed on the surface of the megaspores. The frequency of female gametophytes developing from immature megaspores cultured in MS basal liquid medium (pH 7) supplemented with 1 mgl-1 GA3 was 46%. However, these female gametophytes derived from megaspore only culture could not differentiate into sporophytes. The mixed culture of microspores and megaspores resulted in successful sporophyte regeneration. The highest frequency (12.3%) of green sporophyte regeneration from mixed spore culture occurred when the cultures were maintained at 25℃ under cool-white fluorescent light (40 μmol·m-2·s-1) with a 16 h photoperiod. Regenerated sporophytes were transferred to a test tube containing vermiculite and a sand mixture and left there until they had three leaves. After root growth and the fifth leaf had emerged, more than 95% of the regenerated sporophytes were successfully transferred to the soil and grown to mature plants. The sporophyte regeneration system established in this study could be successfully used for the restoration of the endangered aquatic species, I. coreana.

Biological Control of Damping off ( Pythium aphanidermatum ) Using Streptomyces lavendulae Isolate 21 on Tobacco Seedbeds

Soil actinomycetes, particularly Streptomyces spp., possess antagonistic activity against a wide range of plant pathogens as they are potent producers of antibiotics. In the present study 40 isolates of actinomycetes were isolated from tobacco soils of India and screened against Pythium aphanidermatum in vitro . Among them, ten Streptomyces isolates showed high antagonistic activity. Streptomyces lavendulae isolate 21 showed strong inhibition zone (24mm) of the pathogen. Experiments in vivo using spray of mixed spore suspension of P. aphanidermatum and S. lavendulae isolate 21 on tobacco seedbeds resulted in marked reduction of disease severity, significant improvement in dry weight, leaf area and nutrient content. The treatment P. aphanidermatum S. lavendulae recorded 32 per cent increase in number of transplantable seedlings against P. aphanidermatum alone, while the treatment Streptomyces alone recorded 1.87 times increase in number of transplantable seedlings against control.

Biological Control of Rice Blast (Magnaporthe oryzae) by use of Streptomyces sindeneusis isolate 263 in Greenhouse

Soil Actinomycetes particularly Streptomyces spp. have antagonistic activity against wide range of plant pathogens. In the recent decades they have attracted high interests as biocontrol agents. In search for finding such principles, in vitro suppression of Magnaporthe oryzae the causal agent of rice blast disease was studied by use of Streptomyces sindeneusis isolate 263 in greenhouse. Spray of rice seedling-leaves with of mixed spore suspension of the pathogen and S. sindeneusis isolate 263 resulted in strong inhibition of the pathogen and suppression of leaf symptoms. Propagation of the antagonist crude sap was performed in aqueous cultures and bioactivity was monitored in shaked cultures. Ongoing goals of this research include isolation, characterization and identification of the active metabolites and future goals include identification of active genes for use in development of recombinant DNAs in transgenic rice varieties bearing elevated resistance to infections by M. oryzae.