PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds

Abstract

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.     

A survey of die-back disease of neem in Tamil Nadu,India and PCR-based confirmation of the isolates

A survey of die-back disease of neem was done in different agro climatic regions of Tamil Nadu, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die-back were collected from different regions of Tamil Nadu, India and they were further analyzed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die-back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the confirmation of P. azadirachtae – the causative agent of die-back of neem by Polymerase chain reaction (PCR). Studies revealed the amplification of expected 141bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Tamil Nadu confirming the causal organism of die-back of neem. Studies revealed a very high incidence of die-back in most of the places of Tamil Nadu. Hand held GPS was used in the study which would help in continuous monitoring of the diseased trees.     

Study of die back disease incidence of neem in Karnataka,India and PCR based identification of the isolates

A disease survey of die back of neem was done in different agroclimatic regions of Karnataka, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die back were collected from different regions of Karnataka, India and they were further analysed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the detection of P. azadirachtae, the causative agent of die back of neem by polymerase chain reaction (PCR). Studies revealed the amplification of expected 141 bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Karnataka indicating the causal organism of die back disease on neem. Studies revealed a very high incidence of die back in most of the places of Karnataka. This is the first report on disease incidence of die back of neem. Hand held GPS was used in the study which helps in continuous monitoring of the diseased trees.     

In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

Estimation of Different Cellular Compounds from the Mycelium of Phomopsis theae, Causal Agent of Phomopsis Canker Disease of Tea

Variations between three isolates (MP, TP and WP) of Phomopsis theae were studied based on the growth pattern, cellular constituents in the mycelium and protein expression profile. The growth initially increased and later decreased. The growth of these isolates in terms of mycelial dry weight exhibited in the order of MP > TP > WP isolates. The quantitative estimation of cellular constituents such as total sugar, reducing sugar, non‐reducing sugar, nitrogen, protein, amino acids, polyphenol, catechin and lipid showed that there was no significant difference in these constituents between the isolates. The detection of amino acids in the mycelium of P. theae isolates showed 16 free forms and 11 bound forms. Amino acids in the bound form were elucidated to be higher in all the isolates than in the free form. All the isolates of P. theae exhibited prominent protein pattern with three common bands, which lies between the molecular weight of 116 and 66.2 kDa.     

Effect of the stem blight pathogen, Phomopsis emicis, and the weevil, Perapion antiquum, on the weed Emex australis

The stem blight pathogen, Phomopsis emicis, and the weevil, Perapion antiquum, are two potential biological control agents for the annual weed, Emex australis. Neither pathogen nor weevil affected the development of rosette (5-wk-old) E. australis, but both significantly reduced stem length and number of new fruits in mature plants (10-wk-old) in growth room experiments. Stems grew two-thirds less than controls in plants attacked by weevils, had no net increase when both weevils and fungus were present, and collapsed in the presence of the fungus alone. Attack by weevils elicited a host response that slowed the development of the disease. Phomopsis emicis and Perapion antiquum reduced the ability of E. australis to produce new fruits by 77% and 68%, respectively. Pathogen and weevil together reduced the number of new fruits by 83%. Perapion antiquum did not carry or provide infection sites for P. emicis. The lack of damage by pathogen and weevil to rosettes could compromise their effectiveness as biological control agents.     

In vitro evaluation of six fungicides on radial mycelial growth and regrowth of Fusarium pallidoroseum isolated from castor (Ricinus communis) in Samaru,Nigeria

Six fungicides at three rates (1.5x, 1.0x and 0.5x mg a.i/ml) were evaluated on radial growth and and regrowth of mycelia of Fusarium pallidoroseum isolated from castor (Ricinus communis) in vitro. It was observed that the fungicides (Benomyl, Benomyl + Thiram, Mancozeb, Metalaxyl-m + Thiomethoxan + Difenconazol, Tricyclazole and Carbendazim + Mancozeb) at all the concentrations tested inhibited mycelial growth and regrowth of the fungus. Benomyl, Benomyl + Thiram and Tricyclazole completely inhibited mycelia growth of fungus at 1.5x, 1.0x and 0.5x mg a.i/ml. Metalaxyl-m + Thiomethoxan + Difenconazole, Carbendazim + Mancozeb partially inhibited radial growth and re-growth of mycelia only at 1.5x mg a.i/ml, not at 1.0x and 0.5x mg a.i/ml. The inhibitory effect of all the fungicides on mycelia growth and re-growth was greatest at 1.x5 mg a.i/ml.     

In vitro screening of pentamidine analogs against bacterial and fungal strains

A series of linear pentamidine analogs exhibiting low cytotoxicity, active against Pneumocystis carinii, were evaluated for in vitro activities against bacterial and fungal strains. The majority of the tested bis-amidines exhibited marked activities against Gram-positive strains. In view of the fact that the highest potency was found for 1,5-bis(4-amidinophenoxy)-3-thiapentane dihydrochloride 1j with the S atom in the middle of the aliphatic linker, four new pentamidines bearing S atoms were synthesized and also evaluated against MRSA strains. N,N′-Dialkylated pentamidines with S atoms in the linker are the promising lead structures for antimicrobials development.     

In vitro screening of biological and chemical agents together on the growth of Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. causing inflorescence die back in arecanut

Colletotrichum gloeosporioides is a destructive pathogen of many crop species causing diseases in many annual, biennial and perennial plants. A study was undertaken to find out the effect of biological and chemical agents together on the growth of C. gloeosporioides causing inflorescence die back in arecanut at the Department of Plant Pathology, CPCRI, Kasaragod. To reduce the release of chemical pesticides to the environment, integrated control strategies have been adopted extensively by combining both bioagents and chemical agents. So in the present study in vitro experiments were conducted with two compatible Trichoderma sp. viz., Trichoderma virens and Trichoderma viride and fungicides viz. Blitox 50 W and Mixol 72. The results indicated that all the treatments had a significant inhibitory effect on the growth of C. gloeosporioides and reduced its colony diameter. High percent inhibition was found when 0.05% of Mixol 72 was used with T. virens (87.61%). The least inhibition was shown by T. virens+0.05% Blitox 50 W (80.95%). It is concluded that the combination of bioagents with fungicides provided higher disease suppression than achieved with fungicides and bioagents when used alone.     

Effects of two neem insecticide formulations on the attractiveness, acceptability and suitability of diamondback moth larvae to the parasitoid, Diadegma mollipla (Holmgren) (Hym., Ichneumonidae)

Abstract:  Behavioural responses of female Diadegma mollipla to volatiles from cabbage plants and host-infested [ Plutella xylostella (L.)] cabbage plants sprayed with two neem insecticide formulations were investigated in a Y-tube olfactometer. Parasitoids were significantly more attracted to volatiles from cabbage and host-infested cabbage sprayed with the powder formulation than to clean air. In contrast, parasitoid response to volatiles from cabbage and host-infested cabbage sprayed with the oil formulation was not significantly different from clean air. In choice tests between infested plants sprayed with water (control) or the powder formulation, parasitoids showed no preference for volatiles from either of the treatments. In similar tests with the oil formulation, parasitoids showed a preference for volatiles from control plants over plants sprayed with the oil formulation. In host acceptance and suitability tests, parasitism rates in the neem- and water-sprayed hosts were, with one exception, not significantly different. However, the neem-sprayed larvae died earlier than control larvae and were therefore not able to support parasitoid development. The implication of these findings for the combined use of neem insecticides and parasitoids in the management of P. xylostella is discussed.     

Effects of neem oil on feeding behaviour and development of the pear sawfly, Caliroa cerasi

Neem oil deterred feeding by pear sawfly, Caliroa cerasi L., larvae (Hymenoptera: Tenthredinidae), both in choice and in no-choice cherry leaf disk bioassays. Deterrence was greater in the choice tests, with 50% inhibition occurring at 0.49% aqueous neem oil compared with 1.11% in the no-choice tests. Antifeedant activity towards pear sawfly larvae is slightly less than has been observed for Lepidoptera, but is higher than deterrence to other insects such as aphids. Topical application of neem oil to sawfly larvae resulted in reduced feeding, increased mortality, and a trend towards slower development. Most larval mortality after neem treatment resulted from incomplete subsequent moults. The potential utilization of neem insecticides for control of pear sawfly in tree fruit pest management is discussed.     

Effects of the neem product, RD-Repelin, on settling behaviour and transmission of zucchini yellow mosaic virus by the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae)

The botanical product RD-Repelin® was highly repellent to pea aphids at concentrations of 1%, 4% or 10%. Repellency occurred prior to leaf contact by aphids at all three concentrations. RD-Repelin® delayed symptom expression of zucchini yellow mosaic virus (ZYMV) in 81% of plants treated with 1% concentration, although virus transmission was not prevented. The effects of RD-Repelin® on aphid settling behaviour, symptom expression and potential for pest management are discussed.     

Effect of neem on the growth and development of the obliquebanded leafroller, Choristoneura rosaceana

Neem, Azadirachta indica A. Juss. (Meliaceae), seed oil (NSO) added to meridic diet at concentrations as low as 0.016% reduced pupation and prevented adult eclosion of obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). At a rate of 0.0016%, NSO reduced the fitness of C. rosaceana, resulting in longer developmental times, lower adult eclosion rates, and reduced egg production compared with controls. The neem insecticide Margosan-O ^TM produced comparable results based on concentrations of the most biologically active constituent, azadirachtin. Pupation was completely inhibited at concentrations of 0.25% and 1.0% for larvae exposed at 5th or 6th instar, respectively; rates as low as 0.016% reduced pupal weights and adult eclosion rates. For larvae transferred to treated diet at 5th instar, physical abnormalities in the wings of adults occurred at a rate of 0.004% NSO and increased with increasing treatment rates. NSO at concentrations as high as 2.0% was not antifeedant to neonate larvae, based on 24 and 48 h choice test bioassays, when incorporated into a meridic diet.     

In vitro standardisation of resistance screening methods in cassava against tuber rot disease

Cassava tuber rot caused by Phytophthora palmivora in growing regions of Tamil Nadu and Kerala, is causing yield loss up to 80%. In the present study, resistance reactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots by artificial inoculation method in search of a suitable in vitro resistant screening method. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysis exhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analysis was superior in discriminating even small variations in resistance reactions than tuber analysis. The cultivar Sree Padmanabha showed higher resistance than other cultivars and the level of resistance in a cultivar is heritable which could be helpful in breeding programme. Based on the results it can be concluded that leaves of cassava could be used for screening resistance in the host and also in analysing the virulence of the isolate. This is the first report on screening the resistance in cassava cultivars against root rot caused by P. palmivora.     

积雪草漆斑病病原鉴定及室内药剂筛选

近年来,在广西各地积雪草种植基地,出现一种为害极重的病害,该病害病原菌主要为害积雪草叶片、叶柄,可致使叶片大面积枯死,该病害发生普遍、侵染期长,严重影响积雪草药材的品质和产量。该研究采用常规组织分离法从积雪草病叶上分离病原菌并根据柯赫氏法则验证其致病性;通过菌落特征、菌株形态及ITS序列分析鉴定病原菌;在室内利用生长速率法测定10种杀菌剂对该菌的抑制活性。结果表明：为害积雪草叶片的病原菌为泛生漆斑菌( Myrothecium inundatum);该菌侵染性强,接种至健康积雪草叶片后24 h即可发病,并迅速扩展引起叶片萎蔫;杀菌剂60％唑醚?代森联水分散粒剂、45％恶霉灵?甲霜可湿性粉剂、1.8％辛菌胺醋酸盐水剂、80％代森锰锌可湿性粉剂室内对积雪草漆斑病病原菌的抑菌率均在98％以上,抑菌效果极强,建议在生产中轮换应用防治积雪草漆斑病。15％咪鲜胺微乳剂、70％甲基硫菌灵可湿性粉剂和10％苯醚菌酯悬浮剂对该病原菌的抑菌率也均在92％以上,抑菌效果良好,在生产中可酌情考虑使用。     

Inhibition of the growth of apple pollen tubes by EDTA, surfactants and fungicides

The effects of EDTA (ethylene diamine tetraacetic acid), Kresoxim‐methyl, Wettable Sulphur 80 WP and the surfactants Triton X‐100, Triton X‐114, Dehydol TA 5 and Dehydol TA 29 on pollen tube growth of Malus domestica, Borkh. cvs ‘Golden Delicious’ and ‘Mondial Gala’ were determined by image analysis. The ED₅₀ ‐ and ED₉₀ ‐ values (effective doses giving 50% and 90% reduction of growth) of the chemicals, except for the surfactants, were determined. The selected test substances showed an inhibiting effect on pollen tube growth, with Kresoxim‐methyl being the most active. Surfactants are components of pesticides and they also caused inhibition of pollen tube growth but only at application rates more then 10‐fold higher than typically recommended. Pollen of both apple cultivars were almost equally sensitive to the chemicals.     

Novel Fusicoccins R and S,and the Fusicoccin S Aglycon (Phomopsiol) from Phomopsis amygdali Niigata 2-A,and Their Seed Germination-stimulating Activity in the Presence of Abscisic Acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3α-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3′-deacetyl-3α-hydroxyfusicoccin A and 3β-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3α-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3α-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3α- or 3β-hydroxy group in their diterpene moiety are briefly discussed.