Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

芝麻菜品种对尖镰孢菌枯萎病的抗性评价

尖镰孢菌枯萎病是意大利西北部芝麻菜栽培品种(Eruca vesicaria)和野生品种(Diplotaxis spp．)上的重要病害之一,常引起严重的损失。为评价芝麻菜品种对尖镰孢菌枯萎病的抗性,2004年6～11月,选用了44个栽培及野生品种资源在玻璃温室进行抗性试验,使用浓度为1×10~6cfu/ mL的孢子悬浮液蘸根法人工接种30天苗龄的芝麻菜幼苗,以分离自芝麻菜两个不同野生品种的菌株Fus．Ruc．9A/02和Fus．Ruc．13/03,以及分离自栽培和野生品种的32个菌株混合(MIX),分别作为抗病试验接种体,每次接种试验设A、B、C三个重复。整个试验重复进行2次。调查病害引起的死苗率(%)和病情指数(DI),作为评价芝麻菜品种抗性差异的两项指标。研究结果显示,这两项指标间的相关性极显著(P＜0．01),即表明可采用其中一项指标评价芝麻菜品种对尖镰孢菌枯萎病的抗性。供试的44个栽培及野生芝麻菜品种中,多数品种抗尖镰孢茵茵株Fus．Ruc．13/ 03,而对菌株Fus．Ruc．9A/02及菌株混合MIX表现为易感性,而且这些易感品种在菌株Fus．Ruc．9A/02及菌株混合MIX两试验中的抗性表现较为一致,Fus．Ruc．9A/02和MIX的毒性也更强于Fus．Ruc．13/03菌株。芝麻菜品种12/03和品种20/03高抗菌株混合MIX,品种6/03和品种7/04高抗菌株Fus．Ruc．9A/02,而品种9/02、2/03、5/03、6/03、7/03、9/03、11/03、12/03、20/03、21/03、24/03、3/04、6/04、7/04和11/04高抗菌株Fus．Ruc．13/03。     

Molecular characterization of Fusarium oxysporum f.sp. cichorii pathogenic on chicory (Cichorium intybus)

Fusarium oxysporum is a ubiquitous soilborne ascomycete responsible for vascular wilt in many plant species worldwide. This species comprises more than 120 putative host-specific formae speciales capable of causing marked economic losses. In summer 2009, wilt symptoms, including chlorosis and poor development of the root system, were observed on cultivars of chicory (Cichorium intybus) in northern Italy. The causal agent isolated from symptomatic tissues in this case was identified as F. oxysporum on the basis of both morphological features and molecular analyses. In this work, we attempted to characterize the isolates of F. oxysporum from C. intybus by both biological and molecular approaches. Pathogenicity trials performed on five species of the Asteraceae family with isolates of F. oxysporum from C. intybus indicated that the pathogen has a unique host range, infecting chicory only. Neither lettuce nor endive, lawn daisy or Paris daisy developed the disease. Five cultivars within C. intybus species were tested, and the cv. ??Clio?? was the most susceptible. Phylogenetic analyses relative to the ribosomal intergenic spacer (IGS) and translation elongation factor 1-alpha (EF1-??) assigned isolates pathogenic to chicory to a single cluster, distinct from other pathogenic F. oxysporum. In light of these findings, we propose to designate this organism as Fusarium oxysporum f.sp. cichorii.     

Formation of Biogenic Amines and Associated Biochemical and Microbial Attributes of Whole Sutchi Catfish (Pangasianodon hypophthalmus) during Iced Storage

Sutchi catfish (Pangasianodon hypophthalmus) is a highly preferred farmed species that is produced in huge quantities. Meat from ice-stored (4 ± 2°C) whole Sutchi catfish was evaluated for formation of biogenic amines, such as putrescine, cadaverine, histamine (HIM), agmatine, tyramine, spermine, and spermidine, and compared with biochemical, microbial, and sensory attributes during 22 days. Analysis of content of biogenic amines in the meat was carried out by liquid chromatography with tandem mass spectrometry using a without derivatization method. Three amines, namely, tyramine, spermidine, and spermine, were only present on 0^th day of storage. Presence of cadaverine was noticed from 9^th day onwards, where the aerobic plate count (APC) reached 4.85 log CFU/g. Putrescine was detected on the 22^nd day of storage, where the APC crossed 7 log CFU/g. HIM was detected in lower quantities from 3^rd day onwards. A shelf life of 15 days was determined based on sensory and microbiological evaluation. Although the samples were biochemically acceptable throughout the storage period, APC exceeded the limit on day 19, and the gradual increase of H₂S-producing bacteria, Brochothrix thermosphacta, Aeromonas, and Enterobacteriaceae, was observed during storage.     

Expression of kit ligand and insulin‐like growth factor binding protein 3 during in vivo or in vitro development of ovarian follicles in sheep

Expression of Kit ligand (KL) and insulin‐like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs’ isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin‐like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage‐specific patterns of expression of these and other developmentally important genes.     

Effect of different organic amendments on lettuce fusarium wilt and on selected soilborne microorganisms

This study evaluated the effect of different organic amendments on lettuce fusarium wilt caused by Fusarium oxysporum f. sp. lactucae in pots under controlled conditions. Their effects on the density of the pathogen, on the total fungi and on fluorescent Pseudomonas spp. were also evaluated after two subsequent lettuce crops. A significant reduction in the severity of the symptoms of F. oxysporum f. sp. lactucae was found after the use of Brassica carinata pellets (52–79% reduction) and compost (49–67% reduction), while Brassica green manure and cattle and chicken manure only provided partial control of fusarium wilt. However, variations in effectiveness were observed for the same treatment in repeated trials. In general, an increase was observed in Pseudomonas and a decrease in fungal populations in the growing medium, which was obtained by mixing a blonde sphagnum peat and a sandy loam soil with B. carinata pellets and compost after two consecutive cropping cycles. Prolonging the Brassica and compost treatments from 30 to 60 days did not significantly affect disease severity, plant growth or the microbial population of the total fungi or Pseudomonas. The largest lettuce biomass was obtained in the non‐inoculated growing medium amended with brassica flour, chicken manure, B. carinata pellets and compost, as a consequence of fertilization. The treatment with B. juncea green manure, B. carinata (pellets and flour) and compost applied 30 days before planting led to promising results and merits further investigation for use under field conditions.     

Management of downy mildew of sweet basil (Ocimum basilicum) caused by Peronospora belbahrii by means of resistance inducers, fungicides, biocontrol agents and natural products

To control downy mildew of sweet basil (Ocimum basilicum L.), incited by Peronospora belbahrii Thines, a number of compounds were tested in 2011 and 2012 under glasshouse conditions. These included copper-based fungicides, biocontrol agents, and compounds previously reported to induce resistance in plants to pathogens. Results were compared with those provided by fungicides registered for downy mildew control in Italy. The tested compounds were used alone or applied in rotation in spray programs. In all trials, the greatest reduction in disease incidence and severity was found with treatments that included metalaxyl-M?+?copper hydroxide, a mineral fertilizer ‘Alexin’, mandipropanid, and azoxystrobin. The glucohumates activator complex and acibenzolar-S-methyl also provided significant disease control (P?<?0.05). The mineral fertilizer Alexin, the glucohumates activator complex and acibenzolar-S significantly reduced disease incidence and severity 20 days after the last treatment compared with the untreated control (P?<?0.05). Among the copper-based products, the greatest reductions in disease incidence and severity were provided by copper hydroxide with terpenic alcohols and copper oxychloride?+?copper hydroxide. The mineral fertilizer Kendal and prohexadione-Ca, as well as mustard oil, partially reduced disease incidence and severity compared with the untreated control (P?<?0.05), whereas the biocontrol agent Bacillus subtilis QST 713 and thyme oil extract were not effective in two out of three trials. When different combinations of various products used in rotation were tested, effective control was found either using a rotation of fungicides with compounds that can induce resistance, as well as by using rotation with different resistance-inducing compounds on their own.     

Evidence for an expanded host range ofFusarium oxysporum f.sp.raphani

The pathogenicity of four isolates ofFusarium oxysporum obtained from infected cultivated rocket (Eruca vesicaria) and wild (sand) rocket (Diplotaxis tenuifolia) was tested on the following cruciferous hosts: stock, radish, wild and cultivated rockets, and various species in the cabbage tribe: cabbage (Brassica oleracea var.sabauda), cauliflower (Brassica oleracea var.botrytis), Brussels sprouts (Brassica oleracea var.gemmifera), broccoli (Brassica oleracea var.italica), turnip (Brassica rapa var.rapa). The results indicated that isolates ofF. oxysporum from cultivated and wild rocket belong to theforma specialis raphani. The isolates from rocket were pathogenic on cabbage, Brussels sprouts, broccoli, turnip, radish and stock; isolates ofF. oxysporum conglutinans from cabbage and radish, and the isolate ofF. oxysporum f.sp.raphani from rape obtained from the ATCC collection, were pathogenic on both cultivated and wild rocket.     

Effect of antagonistic<Emphasis Type="Italic">Fusarium</Emphasis> spp. and of different commercial biofungicide formulations on Fusarium wilt of lettuce

Five experimental trials were carried out to test different biological control agents against Fusarium wilt of lettuce, cause byFusarium oxysporum f.sp.lactucae. In the presence of a very high disease incidence, the best results in terms of disease control as well as increased growth response were shown byTrichoderma harzianum T 22 (RootShield), which, at 3 gl ⁻¹ of substrate, provided very consistent results.F. oxysporum IF 23 gave good disease control but in two out of five trials reduced the biomass produced. Less consistent, but still significant results were provided byF oxysporium MSA 25, at 3 gl ⁻¹ of substrate, and byTrichoderma viride TV 1. The twoF. oxysporum agents Fo 251/2 and Fo 47 and the mixture ofT. harzianum + T. viride (Remedier) partially reduced disease incidence but were less effective than the above mentioned. Less interesting results were offered byStreptomyces griseoviridis (Mycostop). The results obtained show that biological control can play a role in the management of Fusarium wilt of lettuce.