A novel chitosan biopolymer based Trichoderma delivery system: Storage stability, persistence and bio efficacy against seed and soil borne diseases of oilseed crops.

Management of seed and soil borne fungal plant pathogens using fungal species belonging to the genus Trichoderma is gaining importance. Seed coating with powder based formulations of Trichoderma is most widely adopted by the researchers and farmers as well. Delivery system that leads to good adherence of fungal propagules on seed surface, minimizing the wastage of active ingredient, sustained and timely release during treatment process is very important for effective season long protection. Chitosan-PEG (Polyethylene glycol) (Cts-PEG) blend containing Trichoderma harzianum (Th4d) (Cts-PEG-Th) spores is developed and its storage stability, persistence in soil and bio efficacy against seed and soil borne pathogens of groundnut and safflower crops is studied. The blend was stable without much changes in pH throughout the storage period. Persistence studies conducted for 3 months revealed that Cts-PEG-Th amended soil, Trichoderma got released from polymer film slowly and reached a maximum of log 8 CFUs by 30 days and there after started declining to retain log 6 CFUs at 90 days. In shelf life study, the chitosan blend was able to maintain Trichoderma counts of log 10.0 and log 10.2 over a period of 6 months at storage temperatures of 30 °C and 4 °C, respectively and the antagonistic activity unaffected against three plant pathogens viz. Macrophomina phaseolina, Fusarium oxysporumf. sp.ricini and Aspergillus niger over a period of 6 months of storage. Bio efficacy testing in germination towels and green house pot studies revealed the effectiveness of seed treatment with Cts-PEG-Th blend significantly increasing the germination and seedling vigour and reducing the diseases in groundnut (peanut) and safflower.

Study on the correlation between soil microbial diversity and ambient environmental factors influencing the safflower distribution in Xinjiang.

The effects of soil microbial properties and physiographical factors on safflower distributions in the main safflower plantations of Xinjiang province in China were studied. This study may help determine the basis of the environmental factors for evaluating the geoherbalism of this medicinal plant. The soil microbial biodiversity in the bulk soil and rhizosphere of safflower at different growth stages and from different sampling plots were characterized by analyzing the environmental DNAs in the samples. With general primers targeting the 16S ribosomal DNA for bacteria and the internal transcribed spacer 1 gene for fungi, the study was performed using marker gene amplification coupled with Illumina HiSeq high-throughput sequencing technologies. Correlation analysis and a distance-based redundancy analysis were performed to determine the dominant factors affecting the distribution of the microorganism in safflower soils. A total of 16517 bacterial operational taxonomic units (OTUs) were obtained from all the 108 soil samples of nine safflower sampling plots. At the phylum level, 48 phyla have been identified with Actinobacteria (32.9%) and proteobacteria (28.7%) being predominant. For fungi, 8746 OTUs were obtained, which belonged to seven phyla with Ascomycota overwhelmingly superior in relative abundance. A significant positive correlation was found between soil microbe quantity and ASL (above sea level). Safflower was sensitive to changes in elevation, growing more abundantly in the mountainous regions at heights of around 1,200 m above sea level. It is concluded that the dominant factors affecting the distribution of microorganisms in safflower soils were soil moisture, available N, and ASL.

Nested PCR assay for specific and sensitive detection of Alternaria carthami.

Alternaria leaf spot (ALS) caused by Alternaria carthami Chowdhary is one of the major threats to the cultivation of safflower in the world. The pathogen is seed borne and requires early detection for restricting its transmission and proliferation. A PCR-based diagnostic assay was developed for easy, quick and reliable detection of A. carthami in infected seeds and leaf samples of safflower. A primer set, AcSPF and AcSPR was designed using ribosomal internal transcribed spacer regions of A. carthami that consistently produced a distinct amplicon of 340 bp with DNA extracted from thirty A. carthami isolates. The specificity of the primer was confirmed using strains of 26 other strains of Alternaria and four other fungal pathogens of safflower. The sensitivity of detection was further enhanced from concentration of 100 pg by simple PCR to as low as 10 pg fungal DNA by a nested PCR assay using ITS and AcSPF and AcSPR primers. The primer pair also facilitated detection of A. carthami in infected seeds and leaf samples. The study provides an accurate and sensitive diagnostic tool for detection of A. carthami.

Quick and accurate detection of Fusarium oxysporum f. sp. carthami in host tissue and soil using conventional and real-time PCR assay.

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40-80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.

Population structure and virulence analysis of Alternaria carthami isolates of India using ISSR and SSR markers.

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei's Genetic diversity index; h = 0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei's genetic diversity index as h = 0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.

Improvement of safflower oil quality for biodiesel production by integrated application of PGPR under reduced amount of NP fertilizers.

Safflower is an important industrial oil seed and bioenergy crop in semi-arid subtropical regions due to its potential to grow on marginal land and having good percentage of seed oil contents which is an important parameter for biofuel production. However, it is an ignored crop in Pakistan. In order to improve the crop productivity and reduce the use of agrochemicals for sustainable biodiesel feedstock production, an experiment was conducted for two years to improve the fatty acid composition and oil quality of Carthamus tinctorius L. (safflower) by the inoculation of Azospirillum and Azotobacter alone as well as in combined application with nitrogen and phosphate (NP) fertilizers on cultivars Thori and Saif-32 under field conditions. Separation and quantification of fatty acids were done on precise comprehensive two-dimensional gas chromatography (GC×GC). The results showed that fatty acid profile specifically monounsaturated fatty acids i-e oleic acid (C18:1) was significantly improved by Azospirillum supplemented with the quarter dose of NP fertilizers (SPQ) with concomitant decrease in polyunsaturated fatty acids by the respective treatment. Oil quality attributes such as acid value, saponification number, iodine value, refractive index and free fatty acid contents were reduced by the application of Azotobacter and Azospirillum in combination with half and quarter doses of NP fertilizers treatments (BTH, SPH, BTQ and SPQ). The reduction in these variables is positively linked with improved biodiesel yield and quality. It can be concluded that application of Azospirillum and Azotobacter not only reduced the use of NP fertilizers up to 50%-75% but also improved the oil quality in order to obtain environment friendly, sustainable and green fuel.

Effects of harvest date, wilting and inoculation on yield and forage quality of ensiling safflower (Carthamus tinctorius L.) biomass.

BACKGROUND: Safflower (Carthamus tinctorius L.), usually grown as a source of oil crop, can be used as fodder either for hay or ensiling purposes, particularly in semi-arid regions. RESULTS: A 2-year trial was conducted in southern Italy to evaluate the production and forage quality of safflower biomass cv. Centennial, harvested at three different stages: 1, at complete appearance of primary buds (PB); 2, at complete appearance of secondary and tertiary buds (STB); and 3, at 25% of flowering stage (FS). For each stage of growth, 50% of the biomass was ensiled in 4 L glass jars without and with inoculation (Lactobacillus plantarum, LAB), and the other 50% was field wilted for 24 h before ensiling. Dry matter (DM) content and yield (DMY), pH, buffering capacity (BC) and water soluble carbohydrates (WSC) were determined on fresh forage. On safflower silages were also evaluated ammonia-N, crude protein (CP), fibre fractions, fat, lactic and acetic acids, Ca and P, and gas losses. DMY ranged from 4.5 t ha(-1) (PB harvesting) to 11.6 t ha(-1) (FS harvesting). DM content varied from 129 g kg(-1) (PB not wilted) to 630 g kg(-1) (FS wilted). The WSC in forage before ensiling with not wilting ranged from 128 (PB stage) to 105 and 100 g kg(-1) DM at STB and FS stages, respectively. The wilted safflower forage showed a lower WSC compared to wilted forage. The high sugar substrate allowed lactic acid fermentation and a good conservation quality in all the harvesting stages. Silages quality was strongly influenced by the treatment performed. Wilting practice increased DM, pH and NDF contents but reduced lactic acid, acetic acid and NH(3) -N values. Inoculation reduced DM, pH and NDF contents, but increased lactic and acetic acids, CP and ash. CONCLUSION: As result, wilting the forage for 1 day was very effective in the early harvesting stage because this practice significantly increased DM, reducing on the same time the intensive fermentation and proteolysis processes of silage. When harvesting is performed at the beginning of the flowering stage wilting is not necessary.

[Enhancement of thrombolytic activities of Carthamus tinctorius processed with fermentation with a bacillus sp. C2-13].

A processing method to enhance thrombolytic effect of Carthamus tinctorius using a fermentation technology with bacillus sp. C2-13 was investigated. The fibrinolysis and anticoagulation activity of thrombolytic extracts from an optimized fermentation process was studied using a carrageenan induced mice model. The fermented extracts resulted in significantly better thrombolytic activity, suggesting that the process was promising for use in the study and preparation of nature medicines.