Tip Culture Method Research Articles

Efficient Elimination of Actinidia Chlorotic Ringspot-Associated Virus from Infected Kiwifruit Shoots Cultured In Vitro

In this study, methods of Actinidia chlorotic ringspot-associated virus (AcCRaV) elimination by shoot tip culture, thermotherapy followed by shoot tip culture, and chemotherapy followed by shoot tip culture were explored. The results showed that the AcCRaV elimination rate was 23.3% when the secondary shoot tip culture method was used and when the shoot tip length was less than 0.5 mm. The AcCRaV elimination rate was 100% when thermotherapy (36°C [day] and 32°C [night]) was applied for 20 days followed by shoot tip culture (shoot tip length less than 1.0 mm). When shoot segments were treated with ribavirin at 15 µg/ml for 2 months followed by shoot tip culture, the elimination rate of AcCRaV was 100% (shoot tip length less than 1.0 mm). When shoot segments were treated with ribavirin at 25 µg/ml for 2 months followed by shoot tip culture, the elimination rate of AcCRaV was 100% (shoot tip length less than 1.5 mm). This is the first report on kiwifruit virus elimination methods.

GIS-Based Remote Sensing Image Information Analysis of the South Atlantic Ecological Environment and Diversity of Deep-Sea Microorganism with Antifungal Effects

This paper uses the GIS technology to collect location and geographic information of the South Atlantic ecological environment and then uses the remote sensing image technology to extract and analyse the collected information to provide a strong basis for maintaining a healthy marine ecological environment. Based on multitemporal remote sensing images, this paper constructs an ecological vulnerability evaluation model for marine by coupling several ecological indicators, analyses the status of marine ecological environment and its dynamic changes, explores the driving factors of its changes, further analyses the relationship between ecological vulnerability, land use changes, and landscape patterns, and proposes targeted ecological protection countermeasures for marine ecological environment. This paper analyses and studies the quality control mechanism of marine environment monitoring data, the construction of marine environment evaluation model, and the automation of evaluation and early warning response based on the application of GIS, with the support of marine environment evaluation and early warning business, to enhance the availability and integrity of marine environment monitoring data, improve the execution efficiency of marine environment evaluation business and early warning response, reduce the risk of marine environment emergencies, and realize the integration and intelligence of marine environment evaluation and early warning, to improve the level of Informa ionization and management of marine environment evaluation business in marine management departments. The operational watershed ecological environment monitoring service system built in this paper, which integrates GIS, RS, GPS, data warehouse, and other information technologies, has the advantages of high accuracy of information extraction, convenient data management, good portability, and strong operability, which can provide a scientific basis for the unified scheduling management of the marine environment, ecological environment evaluation, and sustainable development planning of the economy, and it is of great theoretical and practical significance and can be used in other watersheds. Meanwhile, the unique environment of the ocean has endowed the marine microorganism with affluent diversity, which can display a variety of bioactivities. In this study, thirty-two seawater and sediment samples were collected from different depths within the scope of 5 m to 3203 m at 18 sampling sites in the South Atlantic Ocean. 1017 culturable strains were obtained after isolation and identification. The tip culture method was used to screen strains with antifungal and antiaflatoxigenic activities. The tip culture results showed that a total of thirty-one marine strains were confirmed to have significant antagonism against aflatoxin production with an inhibition ratio exceeding 80%, including 11 strains with a mycelial growth inhibition ratio exceeding 80% simultaneously. The variability of culturable bacterial distribution and diversity were studied by phylogenetic analysis of 16S rRNA sequences. Phylogenetic analysis of 16S rRNA sequences revealed a significant diversity of culturable strains. A total of 31 strains clustered into 13 genera: Marinobacter sp., Pseudomonas sp., Alteromonas sp., Idiomarina sp., Halomonas sp., Alcanivorax sp., Thalassospira sp., Jannaschia sp., Stakelama sp., Erythrobacter sp., Chengkuizengella sp., Bacillus sp., and Microbacterium sp. The 31 strains with antifungal and antiaflatoxigenic activities were dominated by populations of strains belonging to the genus Thalassospira (19.35%). To the best of our knowledge, this is also the first report to analyse the diversity of culturable bacteria with antifungal and antiaflatoxigenic activities in the South Atlantic Ocean from three perspectives: different media, different sampling stations, and different sampling depths.

Fumonisin-production by Aspergillus section Nigri isolates from Japanese Foods and Environments.

Fumonisins are well known as mycotoxins produced by various Fusarium species. Recently Aspergillus niger has been reported to be a fumonisin B2 (FB2) producer. Aspergillus niger is a member of Aspergillus section Nigri. Members of this section are common food contaminants and are also distributed widely in the environment. This study aimed to determine 1) optimum culture conditions of A. niger for fumonisin production including growth medium, temperature and incubation period and 2) fumonisin production among isolates of Aspergillus section Nigri and closely related species isolated from Japanese food and environmental samples. Growth on Czapek yeast extract broth +5% NaCl (CYBS) at 28°C for 7 days resulted in the highest levels of FB2 production as determined by quantitative LC-MS/MS of culture extracts. Sixty-two isolates were collected from various foods in domestic markets as well as from soil and air. The isolates principally separated into two groups; A. niger and A. luchuensis/A. piperis/A. tubingensis, following molecular phylogenetic analysis. ELISA using the tip culture method was shown to be suitable for screening of the fumonisin-producing strains. Phylogenic analysis of Aspergillus section Nigri isolates from food and environmental samples indicated that fumonisin producing strains could be grouped into the A. niger clade. Nineteen of 35 (54%) isolates classified as A. niger were FB2 producers. The current study suggests that FB2-producing A. niger are distributed throughout several regions of Japan.

The value of differential time to positivity of blood cultures in diagnosis of catheter-related bloodstream infection in patients with solid tumors in intensive care unit

To determine the value of differential time to positivity (DTTP) of blood culture for the diagnosis of catheter-related bloodstream infection (CRBSI) in patients with solid tumors in intensive care unit (ICU). A retrospective study was conducted. 615 pairs of peripheral vein blood cultures and instantaneous catheter tip blood culture of 615 patients admitted to ICU of Tianjin Medical University Cancer Institute and Hospital were collected from August 2011 to March 2014. The DTTP method and (or) semi quantitative culture of catheter tip were compared. CRBSI was diagnosed when both cultures were positive for the same microorganism and DTTP≥2 hours (120 minutes). The result of this procedure was compared with that of organism obtained using the semi quantitative culture of blood at catheter tip with ≥15 cfu. Based on the clinical diagnosis, the reliability of two kinds of laboratory examination was compared for the diagnosis of CRBSI by plotting receiver operator characteristic curve (ROC curve). The result of 615 cases suspected of having CRBSI were analyzed during the study period. Of these, 440 episodes were excluded because cultures were negative for blood obtained through peripheral vein and central vein. Eight episodes were excluded because only peripheral vein blood culture was positive and 57 episodes were excluded because of only central vein blood culture was positive, 68 pairs of blood cultures were excluded due to the presence of multiple catheters and repeated blood withdrawals. Two cases of polymicrobial cultures were excluded from the final analysis due to the difficulty in determining the time of positive result for each individual microorganism. Ten cases in 42 cases of suspected cases of CRBSI were excluded from analysis because catheter was not removed, therefore culture from catheter tip could not be obtained. Using the DTTP method, 14 out of 17 CRBSI cases were diagnosed with DTTP≥120 minutes, while 3 cases were missed; the semi quantitative catheter tip culture was positive in 13 cases, and in 4 cases it was neglected. In 2 cases of CRBSI it was missed by both methods. The area under the ROC curve (AUC) of DTTP, catheter tip culture and the combination method was 0.912, 0.882 and 0.941 for diagnosis of CRBSI, respectively. Validity values for the diagnosis of CRBSI for DTTP were: sensitivity 82.35%, specificity 92.31%, positive predictive value 93.33% and negative predictive value 80.00%, and they were higher than those of the catheter tip culture method only (76.47%, 84.62%, 86.67% and 73.33%). The specificity and positive predictive CRBSI combination of the two methods in the diagnosis value were up to 100%, the sensitivity (88.24%) and negative predictive value (86.67%) was also increased, but no significant differences were found with DTTP method (χ2=0.00, P=1.00; χ2=0.00, P=0.98; χ2=0.00, P=0.98; χ2=0.00, P=0.98). DTTP can be a valid method recommended for CRBSI diagnosis in critically ill patients with acceptable sensitivity, good specificity as well as positive predictive value. DTTP combined with other clinical symptoms can not only avoid unnecessary catheter withdrawal, but it also can help obtain the optimal treatment time and strategy.

Antagonizing Aspergillus parasiticus and Promoting Peanut Growth of Bacillus Isolated from Peanut Geocarposphere Soil

A set of 38 Bacillus strains isolated from peanut geocarposphere soil were screened as potential biological control agent anti-Aspergillus parasiticus. Tip-culture method with rapid and quantitative characteristics was used to determine anti-A. parasiticus activity and the process of isolation could be accelerated with this method. 12 out of 38 Bacillus strains showed high anti-aflatoxin production activity. These 12 Bacillus strains were selected to identify the characteristics of promoting peanuts germination rate. Pot experiment in greenhouse was carried out by using these strains which can promote peanuts germination. Phytohormones in the fermentation broth were also detected as another important reference factor to select the isolates as biological control agent with PGPR features. These Bacillus strains isolated from peanut geocarposphere soil not only had high ability anti-Aspergillus parasiticus, but also promoted peanut growth. Therefore, these Bacillus strains were well adapted to peanut production in the field as biological control agent with plant growth promoting rhizobacteria (PGPR) features.

MASS PROPAGATION OF LUNUMIDELLA Melia dubia USING INVIVO AND INVITRO CULTURE METHODS

Lunumidella (Melia dubia) is a fast growing short rotational tree species which can beharvested in 15-20 yrs. The timber is light and mainly used for ceilling and panelling.However due to the presence of a hard seed coat and limited period of viability planting ofLunumidella has become a problem.Therefore investigations were carried out with the objective of finding suitable propagationtechniques using invivo and invitro culture methods. Shoot tip culture method was carriedout using axillary buds and nodel segments taken from plant materials brought from theDodangolla University farm and kept under the running tap water for 2 hours. Thensurface sterilization was done using 10%. Naocl containing 2 drops of teepol/ 100mlsolution for 10 mts. in sterilized conditions. They were then washed with distilled water 3times for 2 min. each and transferred into Mureshige and skoog medium (MS) containingdifferent concentrations of cytokinins.The best medium to grow Lunumedella invitro was 112 strength Ms Agar medium with thesupplement of 1.0 mg/I SAP.In vivo methodsDifferent pretreatment methods were adopted to overcome the poor germination due to thehard seed coat by using acid treatments, base treatments, heat treatments and mechanicalscarification. Seeds were treated with 98% H2 S04 for different time periods; 1 mt, 5 mts.,IOmts, 15 mts, 30mts) and then soaked in water for 24 hours and planted them in polybags in the plant house.Likewise the same procedure was carried out with base Ca(OH)2 and KOH (ash).The best pretreatment method for Lunumidella was acid pretreatment method. In this casethe germination percentage was 40%.

Potential of chitinolytic Serratia marcescens strain JPP1 for biological control of Aspergillus parasiticus and aflatoxin.

Serratia marcescens strain JPP1 was isolated from peanut hulls in Huai'an city, Jiangsu Province, China. Its potential to inhibit the mycelial growth of Aspergillus parasiticus and the subsequent aflatoxin production was evaluated. The strain JPP1 could produce chitinase to degrade fungal cell walls, which was the main mechanism of strain JPP1 for biocontrol. Scanning electron microscopy of fungi treated with the crude chitinase revealed abnormal morphological changes. While the strain was grown in the peanut hulls-based medium, the chitinase activity reached 7.39 units. RT-PCR analysis showed that the crude chitinase repressed the transcription of genes involved in the aflatoxin gene cluster, such as aflR, aflC (pksL1), and aflO (dmtA) genes. By visual agar plate assay and tip culture method, the strain JPP1 exhibited remarkable inhibitory effect on mycelia growth (antifungal ratio >95%) and subsequent aflatoxin production (antiaflatoxigenic ratio >98%). An in vitro assay with seed coating agent of bacterial suspension showed that strain JPP1 effectively reduced fungal growth and subsequent aflatoxin production on peanut seeds, and its antagonistic effect was superior to the common agricultural fungicide of carbendazim. These characteristics suggest that S. marcescens JPP1 strain could potentially be utilized for the biological control of phytopathogenic fungi and aflatoxin in Chinese peanut main producing areas.

Diversity of culturable root-associated/endophytic bacteria and their chitinolytic and aflatoxin inhibition activity of peanut plant in China

A total of 72 isolates of root-associated/endophytic (RAE) bacteria were isolated from peanut plants grown in the main producing areas of 6 provinces in China. The 16S rRNA gene sequences of these isolates were determined and phylogenetic analyses revealed that 72 isolates belonged to the classes Bacilli (49 isolates) and Gammaproteobacteria (23 isolates). The majority of RAE bacteria in Bacilli belonged to 2 genera, Bacillus and Lysinibacillus (48 and 1) while those in Gammaproteobacteria belonged to the genera Enterobacter, Serratia, Stenotrophomonas, and Pseudomonas (7, 11, 3 and 2 isolates, respectively). This is the first report of Lysinibacillus xylanilyticus isolate as biocontrol agent against AFs. All of the selected RAE bacteria showed inhibitory activities against Aspergillus parasiticus (A. parasiticus) growth and/or aflatoxins (AFs) production by visual agar plate assay and tip culture method. Most of the RAE bacteria strains (96 % strains) were determined to have decreased mycelia growth or AFs production levels by >50 % (p < 0.05). Bacterial isolates were further characterized for chitinolytic activity and 22 strains (30 % strains) of identified RAE bacteria degraded colloidal chitin on the chitin medium plate. Ten selected chitinolytic RAE bacteria were tested for antifungal activity on peanuts and most of them significantly decreased mycelial growth and AFs production levels by >90 %. These results showed a wide distribution of biological control bacteria against AFs in Chinese peanut main producing areas and the selected RAE bacteria could potentially be utilized for the biocontrol of toxicogenic fungi.

Mycotoxin-Producing Ability and Chemotype Diversity of Aspergillus Section Flavi from Soils of Peanut-Growing Regions in Iran

Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9%) were able to produce either CPA or AFB1, while eight of the isolates (15.1%) were non-toxigenic. The amounts of CPA and AFB1 produced by the isolates were reported in the range of 18.2-403.8μg/g and 53.3-7446.3μg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4% were producers of CPA, AFB1 and sclerotia (group I), 13.2% of CPA and AFB1 (group II), 9.4% of AFB1 and sclerotia (group III), 13.2% of AFB1 (group IV), 5.7% of CPA and sclerotia (group V) and 15.1% were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB1 and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.

A survey on distribution and toxigenicity of Aspergillus flavus from indoor and outdoor hospital environments

In the present study, genetic diversity and mycotoxin profiles of Aspergillus flavus isolated from air (indoors and outdoors), levels (surfaces), and soils of five hospitals in Southwest Iran were examined. From a total of 146 Aspergillus colonies, 63 isolates were finally identified as A. flavus by a combination of colony morphology, microscopic criteria, and mycotoxin profiles. No Aspergillus parasiticus was isolated from examined samples. Chromatographic analyses of A. flavus isolates cultured on yeast extract-sucrose broth by tip culture method showed that approximately 10% and 45% of the isolates were able to produce aflatoxin B(1) (AFB(1)) and cyclopiazonic acid (CPA), respectively. Around 40% of the isolates produced sclerotia on Czapek-Dox agar. The isolates were classified into four chemotypes based on the ability to produce AF and CPA that majority of them (55.5%) belonged to chemotype IV comprising non-mycotoxigenic isolates. Random amplified polymorphic DNA (RAPD) profiles generated by a combination of four selected primers were used to assess genetic relatedness of 16 selected toxigenic and non-toxigenic isolates. The resulting dendrogram demonstrated the formation of two separate clusters for the A. flavus comprised both mycotoxigenic and non-toxigenic isolates in a random distribution. The obtained results in this study showed that RAPD profiling is a promising and efficient tool to determine intra-specific genetic variation among A. flavus populations from hospital environments. A. flavus isolates, either toxigenic or non-toxigenic, should be considered as potential threats for hospitalized patients due to their obvious role in the etiology of nosocomial aspergillosis.

Isolation of microorganisms and substances inhibitory to aflatoxin production

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The contamination of crops, feeds, and foods with aflatoxins can have serious effects on the health of humans and animals. Although many studies have been done to develop aflatoxin-control strategies, most are limited in their effectiveness. As part of an effort to develop control procedures, we have devised simple and safe methods that are useful for identifying microorganisms that effectively inhibit aflatoxin production by fungi. These include the microtitre agar plate assay using norsolorinic acid-accumulating mutant fungi, the ultraviolet light photography method using an instant film, the tip culture method, a convenient RNA extraction method for reverse transcription-polymerase chain reaction (RT-PCR) analysis, and other methods. Results of a recent trial have shown that Achromobacter xylosoxidans significantly inhibited aflatoxin production by A. parsiticus, and that the main inhibitory substance produced by the bacterium was cyclo(L-leucyl-L-prolyl). This result confirms that the methods described herein are useful for identifying microorganisms that inhibit aflatoxin production by fungi and could contribute to the development of methods to reduce aflatoxin contamination in commodities.

Cyclo( l -Leucyl- l -Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(L-leucyl-L-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml(-1), as determined by the tip culture method. High concentrations (more than 6.0 mg ml(-1)) of cyclo(L-leucyl-L-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(D-leucyl-D-prolyl) and cyclo(L-valyl-L-prolyl), whereas cyclo(D-prolyl-L-leucyl) and cyclo(L-prolyl-D-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(L-leucyl-L-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production.

Timecourse Changes in Two Different Topological Indices with Seminal Root System Development of Rice.

For the topological analysis of plant root systems, two sets of topological indices have been proposed; the ratios of measured altitude (a) and total external pathlength (Pe) to the expected value of the respective parameter when lateral root branching is assumed to occur randomly {a/E(a), Pe/E(Pe)} and to the maximum value of the respective parameter when all lateral roots emerge from the main root axis (herringbone type) {a/Max (a), Pe/Max (Pe)}. We traced the timecourse changes in these indices with root system development of two rice cultivars grown with the excised root tip culture method, and evaluated their adaptability for the actual complexity of the branching pattern. The a/E (a) and Pe/E (Pe) did not adequately express the progress of complication with root growth and the difference between cultivars in branching pattern because root system size substantially affected the values. On the other hand, changes in a/Max (a) and Pe/Max (Pe) showed close correlation with those in several root developmental characters such as the number and mean branching order of lateral roots (MBO). Thus, we conclude that a/Max(a) and Pe/Max(Pe) are better indices than a/E(a) and Pe/E (Pe) for the topological characterization of morphology in the developmental study of rice root systems.

Isolation and characterization of Aspergillus parasiticus mutants with impaired aflatoxin production by a novel tip culture method.

A convenient procedure consisting of UV photography (K. Yabe, Y. Ando, M. Ito, and N. Terakado, Appl. Environ. Microbiol. 53:230-234, 1987) and a tip culture method has been devised for the isolation and characterization of Aspergillus parasiticus mutants relating to aflatoxin production. With the latter procedure, the production of aflatoxins excreted into the culture medium and precursors in the mycelium were easily measured quantitatively or semiquantitatively. A total of 38 mutants in which the aflatoxigenicity was decreased or lost were obtained by UV radiation; 3 were found to be blocked mutants, which accumulated the aflatoxin precursors versicolorin A or averantin.