Leaf Disc Assay Research Articles

Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance

Banana is an important fruit crop and its yield is hampered by multiple abiotic stress conditions encountered during its growth. The NAC (NAM, ATAF, and CUC) transcription factors are involved in plant response to biotic and abiotic stresses. In the present study, we studied the induction of banana NAC042 transcription factor in drought and high salinity conditions and its overexpression in transgenic banana to improve drought and salinity tolerance. MusaNAC042 expression was positively associated with stress conditions like salinity and drought and it encoded a nuclear localized protein. Transgenic lines of banana cultivar Rasthali overexpressing MusaNAC042 were generated by Agrobacterium-mediated transformation of banana embryogenic cells and T-DNA insertion was confirmed by PCR and Southern blot analysis. Our results using leaf disc assay indicated that transgenic banana lines were able to tolerate drought and high salinity stress better than the control plants and retained higher level of total chlorophyll and lower level of MDA content (malondialdehyde). Transgenic lines analyzed for salinity (250mM NaCl) and drought (Soil gravimetric water content 0.15) tolerance showed higher proline content, better Fv/Fm ratio, and lower levels of MDA content than control suggesting that MusaNAC042 may be involved in responses to higher salinity and drought stresses in banana. Expression of several abiotic stress-related genes like those coding for CBF/DREB, LEA, and WRKY factors was altered in transgenic lines indicating that MusaNAC042 is an efficient modulator of abiotic stress response in banana.

A Rapid and Efficient Method for In Vitro Screening of Taro for Leaf Blight Disease Caused by Phytophthora colocasiae

AbstractTaro leaf blight caused by Phytophthora colocasiae presents the single biggest constraint for taro cultivation globally. To accelerate breeding and selection for disease resistance to leaf blight, it is important to develop bioassays which could differentiate resistant and susceptible cultivars efficiently. In this study, thirty taro accessions and four released cultivars were evaluated for resistance to leaf blight using a modified floating leaf disc assay. A novel method for mass production of P. colocasiae zoospores was developed and used as inoculum for the assay. There were significant (P < 0.05) differences among accessions in their response to P. colocasiae infection in the detached leaf assay. The accessions could be efficiently classified into various resistance groups based on a 0–4 score. Also, the assay results were consistent with the field evaluation scores of taro accessions. Thus, this study reports the development of a rapid, simple and repeatable assay that can be used to screen large numbers of taro cultivars for resistance to P. colocasiae.

Polycyclic Spiro Lignans and Biphenyl Tetrahydrofuranone Lignans from Gymnotheca involucrata

Four rare polycyclic spiro lignans (1-4) and four new biphenyl tetrahydrofuranone lignans (5-8) were isolated from the whole plant of Gymnotheca involucrata. The structures of the new compounds were elucidated on the basis of detailed spectroscopic analysis and the absolute configuration of 1 was confirmed by single crystal X-ray diffraction. Bioassay results showed that compounds 2 and 6 exhibited weak antifungal activity against Uromyces viciae-fabae at 100 ppm in leaf-disc assays, while compound 3 demonstrated moderate insecticidal activity against Diabrotica balteata at 500 ppm in an artificial diet assay.

Expression analysis of MusaNAC68 transcription factor and its functional analysis by overexpression in transgenic banana plants

NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors regulating development and responses to stress. One of NAC proteins from banana is NAC68, and expression analysis indicated its positive association to stress conditions. The 5′-proximal region of MusaNAC68 was isolated and sequence analysis indicated presence of stress related cis-elements and cis-elements involved in auxin-signaling. Expression of MusaNAC68 was maximum in roots and positively correlated with application of α-naphthaleneacetic acid. Nuclear localization of MusaNAC68 was determined by fusion of green-fluorescent protein with MusaNAC68 and transiently overexpressing in banana embryogenic cells. Transgenic lines were marginally taller and displayed more abundant roots than control along with altered expression of auxin-responsive genes like auxin-responsive factors and IAA/Aux (indoleacetic acid-induced protein) genes. Transgenic plants overexpressing MusaNAC68 displayed higher activity of polyphenol oxidase and rapid browning of plant extract. Transgenic lines showed better tolerance to stress induced by NaCl and mannitol and produced more shoot biomass. Leaf disc assay showed that transgenic lines retain more chlorophyll and lower malondialdehyde than control under salinity and drought. Transgenic line constitutively overexpressing MusaNAC68 showed elevated expression of many stress-responsive genes indicating its involvement in salinity and drought tolerance in banana.

The Anti-Phytophthora Effect of Selected Potato-Associated Pseudomonas Strains: From the Laboratory to the Field.

Late blight, caused by the oomycete Phytophthora infestans, is the most devastating disease of potato. In organic farming, late blight is controlled by repeated applications of copper-based products, which negatively impact the environment. To find alternative solutions for late blight management, we have previously isolated a large collection of bacteria from the phyllosphere and the rhizosphere of potatoes. Here we report the antagonistic potential of these strains when co-cultivated with P. infestans as well as with other potato pathogens. We then focused on three Pseudomonas strains and compared their protective impact against late blight to that of well-known biocontrol strains in planta using a high-throughput leaf disk assay with automated picture analysis. When sprayed on the leaves of potatoes in the greenhouse, the strains were able to survive for at least 15 days. Under field conditions, populations decreased faster but all tested strains could still be retrieved after 8 days. The most active strain in vitro, P. chlororaphis R47, was also the best protectant on leaf disks from plants grown in the greenhouse experiment, but its protection potential could not be verified in the field due to unfavorable infection conditions. However, its protective effect against P. infestans in planta, its survival in the phyllosphere as well as its ability to colonize the potato rhizosphere in very high population densities, suggest a potential for field application, e.g., in the form of tuber treatment or leaf spray.

Physiological Basis for Glyphosate Tolerance in Hard Fescue and Perennial Ryegrass Cultivars

ABSTRACTGlyphosate effectively controls susceptible biotypes of numerous weed species, but applications are often injurious to turfgrass. ‘Aurora Gold’ hard fescue (Festuca longifolia Thuill.) and ‘JS‐501’ perennial ryegrass (Lolium perenne L.) have enhanced tolerance to glyphosate compared with traditional varieties, but the mechanisms that convey tolerance are not well understood. The objectives of this research were to investigate the physiological basis for glyphosate tolerance in Aurora Gold hard fescue and JS‐501 perennial ryegrass. In greenhouse experiments, the estimated glyphosate rate required to reduce dry shoot mass 50% from the nontreated shoots at 4 wk after treatment measured >3360, >3360, 1850, and 1310 g acid equivalent (a.e.) ha−1 for Aurora Gold, JS‐501, ‘Manhattan V’ perennial ryegrass, and ‘Stonehenge’ hard fescue, respectively. Absorption of 14C‐glyphosate was similar between hard fescue cultivars and averaged 5% of the applied radioactivity. However, Aurora Gold accumulated 20% less shikimate than Stonehenge when incubated at ≥250 μM of glyphosate in leaf disk assays. JS‐501 perennial ryegrass absorbed less 14C‐glyphosate than Manhattan V (13 vs. 22% of the applied) and accumulated ∼50% less shikimate. All cultivars translocated the majority of absorbed radioactivity out of the treated leaf. Based on these findings, glyphosate has less target‐site inhibition in Aurora Gold compared with a more susceptible hard fescue cultivar. The glyphosate tolerance of JS‐501 perennial ryegrass involves two mechanisms including less absorption and target‐site inhibition from a more susceptible cultivar.

Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress.

One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops.

신규 제초활성 물질 발굴을 위한 메타게놈 스크리닝 방법 연구

메타게놈(metagenome)은 연구실에서 배양이 불가능한 미생물을 포함한 자연계에 존재하는 미생물의 유전자를 직접 연구하는 학문분야이다. 지구상 거의 모든 자연, 인공 환경에서 살고 있는 미생물 DNA를 분리 정제하는 것이 가능하며, 재조합 DNA 기술 등을 이용하여 배양 가능한 숙주미생물에 메타지놈을 클로닝함으로서 메타지놈 라이브러리를 제작할 수 있다. 최근 메타게노믹스를 통하여 자연계에 존재하고 있는 대다수의 미생물들이 실험실에서 배양되지 않았던 이유를 구명할 수 있게 되었고, 그들이 가지고 있는 생태학적인 의미와 역할에 대한 이해와 더불어 점차 그 응용 분야와 범위도 확대되고 있다. 이와 같은 메타게놈의 응용 분야 확대의 한 방안으로 본 연구에서는 새로운 제초활성 물질 및 제초활성 물질 생산에 필요한 유전자를 확보하기 위해 메타게놈 라이브러리를 대상으로 오이 떡잎절편 검정, 미세조류 생장저해 검정, 종자발아 저해 검정의 HTS (high throughput screening) 시스템을 구축하였고, 구축된 시스템으로부터 선발된 최종 단일 클론인 9-G1과 9-G12의 바랭이에 대한 in vivo assay를 통해 본 연구에서 개발한 HTS 시스템의 유효성을 확인 하였다. 후속연구로서 활성단일클론이 만들어내는 제초활성물질의 동정 및 제초활성물질을 합성하는 유전자군에 대한 연구를 수행 중에 있다. Metagenomics is a powerful tool to isolate novel biocatalyst and biomolecules directly from the environmental DNA libraries. Since the metagenomics approach bypasses cultivation of microorganisms, un-cultured microorganisms that are majority of exists can be the richest reservoir for natural products discovery. To discover novel herbicidal substances from soil metagenome, we established three easy, simple and effective high throughput screening methods such as cucumber cotyledon leaf disc assay, microalgae assay and seed germination assay. Employing the methods, we isolated two active single clones (9-G1 and 9-G12) expressing herbicidal activity which whitened leaf discs, inhibited growth of microalgae and inhibited root growth of germinated Arabidopsis seeds. Spraying butanol fraction of the isolated active clones' culture broth led to growth retardation or desiccation of Digitalia sanguinalis (L) Scop. in vivo. These results represent that the screening methods established in this study are useful to screen herbicidal substances from metagenome libraries. Further identifying molecular structure of the herbicidal active substances and analyzing gene clusters encoding synthesis systems for the active substances are in progress.

Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance.

Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2, SOR3, and SOR8) selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands.

Effect of Detergent on the Quantification of Grapevine Downy Mildew Sporangia from Leaf Discs

Grapevine downy mildew (DM), caused by the oomycete Plasmopara viticola (Berk. & Curt.) Berlese & de Toni, is a major disease, especially in humid viticultural areas. Development of resistant cultivars is an important objective for grapevine breeding. To establish a reliable and inexpensive quantitative method to aid in breeding for DM resistance, we improved the method for counting the number of sporangia on leaf discs, and developed a method for counting the number of sporangia in solution. To prevent the loss of DM sporangia from adhesion onto plastic ware, we found as little as 0.01% Tween 20 was effective. On the other hand, this detergent was shown to have a severe inhibitory effect upon DM infection of leaves. We developed a sporangia counting method using dried droplets of DM suspensions, and the method was highly correlated with counting by hemacytometer (R2 > 0.96). The nonparametric Spearman’s rank correlations between visual rating and the number of the sporangia were as high as ρ = 0.82 to 0.91, suggesting that evaluation by the visual rating could provide a good estimate of the sporangia numbers on leaf discs. We established a high-throughput and inexpensive method with acceptable accuracy for DM resistance evaluation based on a leaf disc assay, and our results suggested that visual ratings of infected leaf discs provide a good estimate of sporangia numbers.

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (−)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (−)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(−)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(−)-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (−)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.

A leaf disc assay for determining resistance of taro to Phytophthora colocasiae

Phytophthora colocasiae is a destructive fungal pathogen of taro (Colocasia esculenta) which causes taro leaf blight This disease destroyed Samoas taro export industry and led to local food shortages following introduction of the pathogen in 1993 In support of a Pacific regional taro resistance breeding programme laboratory studies in New Zealand aimed to develop a leaf disc bioassay for identifying taro genotypes with disease tolerance A medium has been developed that will retard senescence of the leaf discs for over 14 days The rate of leaf disc infection and subsequent lesion development varied with incubation temperature leaf age and taro cultivar An understanding of these factors and the variation they cause has resulted in a laboratory leaf disc assay for testing resistance of different taro cultivars to taro leaf blight

Sensitivity of Podosphaera xanthii populations to anti-powdery-mildew fungicides in Spain.

Cucurbit powdery mildew caused by Podosphaera xanthii limits crop production in Spain, where disease control is largely dependent on fungicides. In previous studies, high levels of resistance to QoI and DMI fungicides were documented in south-central Spain. The aim of this study was to investigate the sensitivity of P. xanthii populations to other fungicides and to provide tools for improved disease management. Using a leaf-disc assay, sensitivity to thiophanate-methyl, bupirimate and quinoxyfen of 50 isolates of P. xanthii was analysed to determine discriminatory concentrations between sensitive and resistant isolates. With the exception of thiophanate-methyl, no clearly different groups of isolates could be identified, and as a result, discriminatory concentrations were established on the basis of the maximum fungicide field application rate. Subsequently, a survey of P. xanthii resistance to these fungicides was carried out by testing a collection of 237 isolates obtained during the 2002-2011 cucurbit growing seasons. This analysis revealed very high levels of resistance to thiophanate-methyl (95%). By contrast, no resistance to bupirimate and quinoxyfen was found. Results suggest that thiophanate-methyl has become completely ineffective for controlling cucurbit powdery mildew in Spain. By contrast, bupirimate and quinoxyfen remain as very effective tools for cucurbit powdery mildew management. © 2014 Society of Chemical Industry.

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae).

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.

DEVELOPMENT OF TABLE AND RAISIN GRAPES WITH HIGH ANTHOCYANINS USING A LEAF DISK ASSAY

Anthocyanins are considered an excellent source of antioxidant phytochemicals for health benefits. The majority of wine, table and raisin grapes have anthocyanins only in their colored skin. Anthocyanin content of grapes would be increased if their flesh also contained anthocyanins. C33-30, a seedless female flowered table grape with red skin and clear flesh berries was hybridized with the ?Rubired? wine grape, which has black skin and red flesh berries. The F1 progeny segregated in a 3 colored:1 white skin and 1 red:1 clear flesh ratio for colored berries. The seedling expressing the highest level of anthocyanin in the flesh and skin was similar to ?Rubired?. Seven modified backcross families were created by crossing red flesh seeded F1 selections with seedless red skin table or white skin raisin grapes. Skin color segregated as a single dominant gene as expected. One red flesh parent was heterozygous for skin color and the rest were homozygous. Only seedlings with colored skin had red flesh. Flesh color segregated as a 1 red:1 clear in three of the families or in a 2:1 ratio or higher in four families. One red flesh parent produced an abnormally high percentage (>80%) of red flesh seedlings. Leaf color in the leaf disk assay correlated well with flesh color in the F1 family. Seedlings that developed 0 to 20% anthocyanin in the leaf disk assay had clear flesh with white, red or black skin (32% of BC1 population). However 19% of red flesh seedlings would have been discarded using this selection criterion. Part of this discrepancy is due to the fact that leaves taken too early in their development from greenhouse grown plants did not develop anthocyanin.

Production and evaluation of transgenic sorghum for resistance to stem borer

Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2–3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid l-cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20–30% of damage as compared to 70–80% in non-transformed controls.

Behavioural responses of Frankliniella occidentalis Pergande larvae to methyl jasmonate and cis-jasmone

The larval stages of Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) cause more direct feeding damage to plants than the adults. We, therefore, investigated the behaviour-modifying effects on second instar larvae of two jasmonic acid derivatives. The artificial application of methyl jasmonate and cis-jasmone, both at 1 % concentration, deterred the larvae from settling in a dual choice bean leaf disc assay. We observed a dose-dependent feeding deterrence of both jasmonates and calculated the concentration required to reduce the feeding damage by 50 % relative to the control treatment (FDC50) for each jasmonate. The feeding damage was reduced by the application of cis-jasmone at 1 % concentration, but not by the jasmonates at the respective FDC50 in no-choice leaf disc bioassays. However, significantly more larvae left jasmonate-treated whole potted bean plants by migrating to the soil compared with control plants. Our results may be exploited extending behavioural manipulation by using plant compounds in thrips control programmes to the full lifecycle of the pest. Plant compounds could be used in integrated and biological pest management strategies against F. occidentalis in combination with the application of various above and below ground control measures.

Phenotyping for resistance to the sugarcane aphid Melanaphis sacchari (Hemiptera: Aphididae) in Sorghum bicolor (Poaceae)

The sugarcane aphid Melanaphis sacchari (Zehnt.) has become a serious pest of sorghum, particularly during the post-rainy season in India and East and Southern Africa. Therefore, we tested a number of techniques to screen sorghum genotypes for their resistance to M. sacchari. Infesting the plants with aphid-infested leaf cuttings and covering with a nylon net was effective in screening sorghum genotypes for their resistance to M. sacchari. Sprinkling the plants with aphids (filled in an 0.5 ml eppendorf tube) in the greenhouse was also used to confirm whether the resistance of genotypes selected is less susceptible to the aphids under natural infestation. Nine genotypes (Line 61510, ICSV 12001, ICSV 12002, ICSV 12003, ICSV 12004, ICSV 12005, SLR 41, PU 10-1 and DJ 6514) exhibited moderate levels of resistance to M. sacchari. These genotypes also exhibited a lower rate of aphid multiplication in the clip cage and leaf disc assays. The rates of aphid multiplication were lower on the genotypes IS 21807, IS 40615, IS 40616 and IS 40618 than on the susceptible check, Swarna in the clip cage assay under the field conditions. Also, lower rates of aphid increase were also recorded on IS 21807 and IS 40615 in the leaf disc assay under laboratory conditions. Some of the genotypes that exhibited resistance to aphid damage under field conditions showed comparatively higher rates of aphid increase than the susceptible check, Swarna in the clip cage assay, indicating that antixenosis could be one of the components of resistance to M. sacchari in these genotypes. Therefore, the clip cage assay could be used to gain further understanding of the mechanisms of resistance to M. sacchari. There is a need to assess the role of antixenosis and colonization in genotypic reaction against M. sacchari to identify the lines with different mechanisms of resistance to this pest. The results suggested that the nylon net technique could be used to screen sorghum genotypes for resistance to M. sacchari. The genotypes exhibiting resistance to M. sacchari can be used to develop aphid-resistant sorghums for sustainable crop production.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

In vitro compatibility of microbial agents for simultaneous application to control strawberry powdery mildew (Podosphaera aphanis)

Biological control of plant pathogens on strawberries may be improved by the simultaneous application of different biological control agents (BCAs). Therefore, the compatibility of various BCAs which had previously shown to be effective against powdery mildew (Podosphaera aphanis (Wallr.) U. Braun & S. Takam) under laboratory conditions was examined in vitro. Inhibitory effects between fungal and bacterial BCAs were demonstrated in dual culture tests on two solid nutrient media. Leaf disc assays with single and multiple strain treatments demonstrated either unaffected or significantly improved control of P. aphanis for many multiple strain treatments, even if antagonistic interactions previously occurred in dual culture tests. Highest inhibition of powdery mildew conidiation (80.7% reduction) was achieved with multiple strain treatments with Bacillus subtilis FZB24 and Metarhizium anisopliae (p < 0.001). In this combination, conidiation was 3.7 times lower than in single treatments with B. subtilis indicating synergistic interactions between these BCAs. Combinations of Trichoderma harzianum T58 and B. subtilis FZB24 showed antagonistic interactions in dual culture tests as well as in leaf disc assays. In this combination, powdery mildew conidiation on leaf discs was four times higher compared to single treatments with T. harzianum T58.