Efficacy of Boric Acid,Monopotassium Phosphate and Sodium Metabisulfite on the Control of Apple Scab

In vitro experiments indicated that boric acid, monopotassium phosphate, sodium metabisulfite and synthetic fungicide fluopyram + tebuconazole were effective in inhibiting conidia germination and germ‐tube elongation of Venturia inaequalis. Monopotassium phosphate even at the highest concentration used in the study reduced conidia germination and germ‐tube elongation of V. inaequalis by 22.1% and 28.8%, respectively; however, the difference between two compounds at lower concentrations except 0.05% (for conidia germination) and 0.1% (for germ‐tube elongation) of boric acid was statistically significant (P ≤ 0.05). Complete inhibition was achieved by 0.01% sodium metabisulfite, 0.035% fluopyram + tebuconazole and 0.2% boric acid. Two orchard trials were conducted on the highly susceptible cv. Mutsu to apple scab to ascertain the efficacy of 0.2% boric acid, 0.5% monopotassium phosphate, 0.5% sodium metabisulfite and 0.035% fluopyram + tebuconazole for the control of apple scab. In both 2013 and 2014, except for the applications of monopotassium phosphate and sodium metabisulfite, the applications of boric acid and fluopyram + tebuconazole to trees at 10‐day intervals significantly reduced disease incidence and severity on leaves and fruit compared to the water‐treated control. In both years, the efficacy of boric acid and fluopyram + tebuconazole treatments was similar in reducing both disease incidence and severity on leaves and fruit in all monthly assessments from July to September. All treatments were neither phytotoxic to leaves and fruit nor did they adversely affect quality parameters of harvested fruit. These results show that boric acid treatment may be applied as an alternative chemical for the control of apple scab.     

Activation of Systemic Resistance to Magnaporthe oryzae in Rice by Substances Produced by Fusarium solani Isolated from Soil

Of 70 micro‐organisms (fungi, bacteria and actinomycetes) isolated from soil using vegetable tissue baits, 16 produced substances in culture fluids capable of preventing the development of blast caused by Magnaporthe oryzae on rice leaves with little or no inhibitory effect on the conidial germination of the pathogen. Isolate KS‐F14, which secreted substances capable of activating resistance in untreated leaves, was selected and identified as Fusarium solani. The resistance‐inducing substances were effective at pH values ranging from 5 to 10 and were stable under high temperatures, maintaining approximately the same level of activity even after autoclaving for 20 min. After application, the activated resistance in rice leaves persisted for 14 days. The polar solvent extracts of freeze‐dried KS‐F14 secretions were effective in activating resistance against M. oryzae in rice plants. The non‐polar solvent extracts were also effective, albeit not as effective as the polar solvent extracts, indicating that although the majority of the secreted resistance‐inducing compounds are hydrophilic, some of the compounds are hydrophobic. Treating secretions with cation or anion exchange resins only partially reduced their resistance‐inducing ability, suggesting that the resistance‐inducing components include both charged and non‐charged compounds. The resistance‐inducing compounds produced by F. solani have the potential to be developed into a commercial product for the control of rice blast and possibly other plant diseases.     

Evaluation of the Anti‐Diabetic Activity of Polysaccharide from Cordyceps cicadae in Experimental Diabetic Rats

Cordyceps cicadae is a medicinal fungus used in treating night sweat, childhood convulsions, vision improvement and pain. This study was designed to evaluate the anti‐diabetic activity of the crude polysaccharide (SHF) from the mycelium and body portion of C. cicadae. Diabetes mellitus was induced in the rat with a single intravenous injection of alloxan monohydrate (150 mg/kg). In other to evaluate the anti‐diabetic effects of C. cicadae polysaccharide in alloxan‐induced diabetic rats, the crude polysaccharide (SHF at 100, 200 and 400 mg/kg body weight) and glibenclamide were administered orally to diabetic rats for 30 days. Blood glucose level, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP), creatinine (CREA), urea, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH) were determined. SHF showed significant reduction in blood glucose in diabetic rats. Treatment of diabetic rats also resulted an improvement in body weights, increased HDL, SOD and GSH, as well as decreased TC, TG, LDL, MDA, urea, CREA, ALT, AST and ALP. These results suggested that C. cicadae polysaccharide displayed anti‐hyperglycemic, anti‐hyperlipidemic and antioxidant activities and could be a promising therapeutic source in managing diabetes mellitus and its associated complications.     

Sequence characterized amplified polymorphic markers for the pitch canker pathogen,Fusarium circinatum

Fusarium circinatum is the causal agent of pitch canker disease of pines. This pathogen is thought to have originated in Central America and currently poses a serious threat to commercial pine plantations in many areas of the world. In this study, polymorphic molecular markers were developed for F. circinatum using the internal short sequence repeats‐polymerase chain reaction (ISSR‐PCR) technique. Nine sequence characterized amplified polymorphic markers were developed. Thirty‐two putative alleles were observed among 103 F. circinatum isolates from different geographical areas using the nine polymorphic markers. These sequence characterized amplified polymorphic markers can be used as genetic tools in populations studies of F. circinatum.     

Two New Anti‐Proliferative C18‐Norditerpenes from the Roots of Podocarpus macrophyllus

Activity‐guided fractionation strategy was used to investigate chemical constituents from the roots of Podocarpus macrophyllus. Successfully, two new norditerpenes, 2β‐hydroxymakilactone A ( 1 ) and 3β‐hydroxymakilactone A ( 2 ), along with ten known analogues ( 3  –  12 ) were isolated. The structures of 1 and 2 were elucidated by spectroscopic analysis including 1D‐, 2D‐NMR, and HR‐ESI‐MS data. The previously reported structure of 2,3‐dihydro‐2α‐hydroxypodolide was revised as 2,3‐dihydro‐2β‐hydroxypodolide ( 3 ) by spectroscopic analysis, and was further confirmed by X‐ray crystallographic analysis. Cytotoxic activities of all isolated compounds against five human solid tumour cell lines (AGS, HeLa, MDA‐MB‐231, HepG‐2, and PANC‐1) were evaluated. All of them exhibited anti‐proliferative activities (IC₅₀ = 0.3 – 27 μm ), except for 10 . Compounds 1 , 4 , 5 , 6 , and 8 exhibited potent inhibitory activities with IC₅₀ < 1 μm against HeLa and AGS cells.     

Fusarium langsethiae – a HT‐2 and T‐2 Toxins Producer that Needs More Attention

Fusarium langsethiae is a toxigenic fungus that was formally described as a new species in 2004. This fungus was first detailed in the 1990s but was initially referred to as ‘powdery Fusarium poae’ having a spore morphology similar to F. poae but a mycotoxin profile like that of Fusarium sporotrichioides. The species has been isolated from infected oat, wheat and barley grains but has been reported as more problematic in the former crop rather than the latter two. Whilst the epidemiology of F. langsethiae remains unclear, the fungus has been shown to produce high levels of type‐A trichothecenes HT‐2 and T‐2 toxins in small‐grain cereals. HT‐2 and T‐2 toxins are two of the most potent trichothecenes capable of inhibiting protein synthesis in eukaryotes. In this regard, mycotoxin contamination caused by F. langsethiae is clearly a food and feed safety hazard. With the European Commission considering legislation of HT‐2 and T‐2 toxins, more information is required not only on the producer and conditions favouring mycotoxin production, but also on reliable methods of pathogen detection and reduction of cereal contamination. This review describes recent research concerning the known epidemiology of F. langsethiae and suggestions of what needs to be known about the fungus in order to be able to understand and employ measures for preventing its infection and contamination of cereals with HT‐2 and T‐2 toxins.     

Modification,Biological Evaluation and SAR Studies of Novel 1H‐Pyrazol Derivatives Containing N,N′‐Disubstituted Urea Moiety as Potential Anti‐melanoma Agents

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF‐mutant melanoma. In continuation of our earlier studies on anti‐melanoma agents based on 1H‐pyrazole skeleton, two sets of novel compounds that include 1H‐pyrazole‐4‐amines FA 1 – FA13 and corresponding urea derivatives FN 1 – FN13 have been synthesized and evaluated for their BRAF^V600E inhibitory and antiproliferation activities. Compound FN 10 displayed the most potent biological activity against BRAF^V600E (IC₅₀ = 0.066 μm ) and the A375 human melanoma cell line (GI₅₀ = 0.81 μm ), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H‐pyrazole‐3‐amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole‐based anti‐melanoma agents.     

Exploratory Studies on the in Vitro Anti‐inflammatory Potential of Two Herbal Teas (Annona muricata L. and Jasminum grandiflorum L.), and Relation with Their Phenolic Composition

The need of new anti‐inflammatory drugs has led to the search for safer and more potent molecules in distinct sources, such as natural products. This work aimed to explore the anti‐inflammatory potential of aqueous extracts from two herbal teas (Annona muricata L. and Jasminum grandiflorum L.) in RAW 264.7 macrophages cells and in cell‐free assays. Furthermore, the phenolic composition of both extracts and of their hydrolysates was characterized by HPLC‐DAD, in order to establish possible relationships with the biological activity. In a general way, A. muricata displayed a stronger capacity to inhibit nitric oxide (NO) production and the activity of phospholipase A₂ (PLA₂), displaying an IC₅₀ value of 142 μg/ml against this enzyme. A deeper look at phenolic compounds revealed that aglycones had more capacity to inhibit NO and PLA₂ than their corresponding glycosides, quercetin being clearly the most potent one (IC₅₀ = 7.47 and 1.36 μm , respectively). In addition, 5‐O‐caffeoylquinic acid, at 1.56 μm , could also inhibit PLA₂ (ca. 35%). Our findings suggest that the consumption of both herbal teas may be a preventive approach to inflammatory disorders.     

Three New Indole Diketopiperazine Alkaloids from Aspergillus ochraceus

Asperochramides A – D ( 1  –  4 ), a new natural product and three new indole diketopiperazine alkaloids, along with seven known analogs ( 5  –  11 ), were isolated from the ethyl acetate extract of Aspergillus ochraceus. Their structures were elucidated by extensive spectroscopic analyses, ECD calculation, and single‐crystal X‐ray diffraction analysis. Compounds 3 and 4 represent a rare group of indole diketopiperazine alkaloid with a 3‐hydroxyl‐2‐indolone moiety. The in vitro anti‐inflammatory effects of compounds 1 and 3  –  11 were investigated by using LPS‐stimulated murine macrophage RAW 264.7 cells. Compounds 1 , 8 , 10 , and 11 showed potential anti‐inflammatory activities.     

Synergistic antimicrobial properties of active molecular chitosan with EDTA‐divalent metal ion compounds

The aim of this study was to elucidate the synergistic antimicrobial activity of chitosan hydrolysates derived from hydrolysis of high molecular weight chitosan (HMWC) with various kinds of divalent metal ions‐EDTA compounds against Penicillium italicum. Amongst hydrolysates, active molecular chitosan (AMC) ranging of molecular weight approximately from 0.8 to 3.0 kDa has been newly researched and demonstrated that it has great antimicrobial activity. Copper, manganese, magnesium, cobalt and zinc ions were used to manufacture chelates with EDTA using a microwave apparatus. Inhibitory effect of AMC or chelates on growth of P. italicum was investigated for up to 72 hr. As the results, AMC showed the highest growth inhibitory activity in the presence of Zn‐EDTA compound compared to the other ion chelates. According to these results, we demonstrated that although Zn‐EDTA‐chelating ligand alone does seem to have a little antimicrobial activity against P. italicum, however, it has synergistic effect against the fungal species when it combined with AMC. Taken together, we suggest that AMC‐EDTA‐Zinc combination (AEM‐Zn) can be a great candidate as natural pesticide for defending postharvest decay of citrus fruits by the fungi.     

Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Antimicrobial Activity of Scabiosa arenaria Forssk. Extracts and Pure Compounds Using Bioguided Fractionation

The emergence of multidrug resistant pathogens threatened the clinical efficacy of many existing antibiotics. This situation has been recognized globally as a serious concern and justifies further research to discover antimicrobial agents from natural origins including plant extracts. The aim of our work was to evaluate the antimicrobial activities of Scabiosa arenaria Forssk . extracts and pure compounds using a bioguided fractionation, and try to explain some traditional use of this genus. The best antimicrobial activity‐guided fractionation was obtained by BuOH fractions of flowers, fruits and (stems and leaves) against Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory concentration (MIC) values from 0.0195 to 5 mg/ml. Escherichia coli was the most affected bug, thus the MIC of fruits BuOH extract showed the best anti‐Escherichia coli activity (MIC = 0.0195 mg/ml), followed by the (stems and leaves) and flowers BuOH extracts; MIC = 0.078 and 0.15 mg/ml, respectively. Furthermore, the subfractions obtained from these three mixed fractions showed also an important antimicrobial activity against the three microorganisms, with MIC values between 0.0195 and 0.312 mg/ml. The fractionation of the aerial part BuOH fraction led to the isolation of oleanolic acid ( 1 ) and luteolin 7‐O‐glucopyranoside ( 2 ) which are reported here for the first time from S. arenaria. Both compounds showed good antimicrobial activities with MIC values ranging from 170 to 683 μm and 86 to 347 μm , respectively. These results support the use of the Scabiosa genus to inhibit the growth of tested pathogenic bacteria and yeasts which may reduce illnesses associated with their exposure.     

Anti‐feeding and insecticidal efficacy of a topical administration of dinotefuran–pyriproxyfen–permethrin spot‐on (Vectra® 3D) on mice against Stegomyia albopicta (= Aedes albopictus)

An ectoparasiticide combining three active ingredients [dinotefuran, permethrin and pyriproxyfen (DPP)] was used in mice in an experiment designed to evaluate its anti‐feeding and insecticidal efficacy against Stegomyia albopicta (= Aedes albopictus) (Diptera: Culicidae) mosquitoes. Twenty‐two adult mice were randomly allocated into two groups consisting of an untreated control group and a DPP‐treated group. Mice were exposed individually for 1 h to a mean ± standard deviation of 27 ± 2 starved female mosquitoes on days 1, 7, 14, 21 and 28 post‐treatment. At the end of the exposure (1 h), mosquitoes were assessed for immediate survival and engorgement status. Additionally, live mosquitoes in both groups were incubated separately and observed for mortality at 24 h after the end of the exposure. The anti‐feeding efficacy of DPP after the 1‐h exposure period was 99.2, 100, 98.0, 89.3 and 87.4% at 1, 7, 14, 21 and 28 days, respectively. Levels of insecticidal efficacy evaluated at 1 h and 24 h after exposure on days 1, 7, 14, 21 and 28 were 36.7, 28.9, 30.8, 23.1 and 11.9%, and 68.4, 45.0, 43.3, 37.9 and 19.9%, respectively. Based on the mouse model, the present study demonstrates that the DPP combination has significant anti‐feeding and insecticidal efficacy against S. albopicta for at least 4 weeks.     

Physicochemical Properties,Antioxidant and Anti‐proliferative Capacities of Dried Leaf and Its Extract from Xao tam phan (Paramignya trimera)

Xao tam phan (Paramignya trimera) has been used for the treatment of cancer and cancer‐like aliments. Among different parts of the P. trimera plant, leaf is considered as a residual part after harvesting of the root. This study aimed to determine the physiochemical properties and the antioxidant and anti‐proliferative capacities of P. trimera leaf (PTL) using microwave drying for the preparation of dry sample; MeOH and microwave‐assisted extraction for the preparation of crude extract; and freeze‐drying for the preparation of powdered extract. The results showed that total phenolic, total flavonoid, proanthocyanidin, and saponin contents of PTL prepared by microwave drying at 450 W were 25.4 mg gallic acid equiv. (GAE), 86.3 mg rutin equiv. (RE), 5.6 mg catechin equiv. (CE), and 702.1 mg escin equiv. (EE) per gram dried sample, respectively. Gallic acid, protocatechuic acid, ellagic acid, rutin, and quercetin were identified in the PTL MeOH extract. Dried PTL displayed potent antioxidant activity, while the powdered PTL extract exhibited great anti‐proliferative capacity on various cancer cell lines including MiaPaCa‐2 (pancreas), HT29 (colon), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2‐C (neuroblastoma), MCF‐7 (breast), MCF‐10A (normal breast), and U87, SJ‐G2, and SMA (glioblastoma). Anti‐proliferative capacity on pancreatic cancer cells (MiaCaPa2, BxPc3, and CFPAC1) of PTL extract (200 μg/ml) was significantly higher (P < 0.05) than those of ostruthin (20 μg/ml) and gemcitabine (50 nm ), and to be comparable to the powdered P. trimera root extract and a saponin‐enriched extract from quillajia bark (a commercial product). The findings from this study allow us to conclude that the PTL is a rich source of phytochemicals that possess promising antioxidant and anti‐proliferative activities, therefore it shows potential as lead compounds for application in the nutraceutical, medicinal and pharmaceutical industries.     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

The Lactoperoxidase System: a Natural Biochemical Biocontrol Agent for Pre‐ and Postharvest Applications

Controlling pests in pre‐ and postharvest crops using natural and low‐impact products is a major challenge. The lactoperoxidase system is an enzymatic system that exists in all external secretions in mammals and is part of the non‐immune system. We tested its efficacy in in vitro microplates on Phytophthora infestans, Penicillium digitatum, Penicillium italicum, Penicillium expansum and Botrytis cinerea to determine the most suitable concentrations for use. Then, we verified its efficacy in planta under controlled conditions. Solutions prepared with 5.4 mm iodide and 1.2 mm thiocyanate and diluted threefold inhibited pathogen growth in vitro by 63–100%. Twofold‐diluted solutions protected potato plants against P. infestans by 60–74% under controlled conditions. Undiluted solution inhibited orange's and apple's postharvest pathogens in curative application with efficacy levels ranging between 84 and 95% in orange and between 63 and 74% in apple. 1.5‐fold concentrated solutions inhibited postharvest pathogens of apple in curative application with efficacy levels ranging between 84 and 92%. Our results also show that the oxidative stress response of fruit following wounding could interfere with ion efficiency. Our tests demonstrate for the first time that this biochemical method is as efficient as a conventional synthetic chemical method under controlled conditions.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.