Participation of the phosphoinositide metabolism in the hypersensitive response of Citrus limon against Alternaria alternata

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata.     

Signal transduction in lemon seedlings in the hypersensitive response against Alternaria alternata: participation of calmodulin,G-protein and protein kinases

The development of an effective hypersensitive response (HR) in any plant system relies, not only in their gene composition and expression, but also on an effective and rapid signal transduction system. Lemon seedlings induce the phenylpropanoid pathway, which results in the de novo biosynthesis of the phytoalexin scoparone, as part of the hypersensitive response against Alternaria alternata. In order to elucidate some of the signaling elements that participate in the development of HR in lemon seedlings, we used several compounds that are known as activators or inhibitors of signal transduction elements in plants or in animal cells. Lemon seedlings treated either with cholera toxin or with phorbol 12-myristate 13-acetate (PMA), in the absence of A. alternata induced phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone, suggesting the participation of a G-protein and of a serine/threonine kinase, respectively, in signal transduction. The use of trifluoperazine (TFP), W-7, staurosporine, lavendustin A or 2,5-dihydroximethyl cinnamate (DHMC) prevented PAL induction as well as scoparone biosynthesis in response to the fungal inoculation, thus allowing us to infer the participation of Calmodulin (CaM), of serine/threonine and of tyrosine protein kinases (TPK) for signal transduction in Citrus limon in response to A. alternata.     

IP3 production in the hypersensitive response of lemon seedlings against Alternaria alternata involves active protein tyrosine kinases but not a G-protein

IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR) of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK) are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC) not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.     

The Response of Citrus limon Seedlings to a Symbiont,Glomus etunicatus,and a Pathogen,Radopholus similis

The influences of a vesicular-arbuscular mycorrhiza (Glomus etunicatus) and burrowing nematode (Radophohts similis), alone and in combination, on the growth of rough lemon (Citrus limon) seedlings were studied in the greenhouse. Growth of mycorrhizal seedlings was significantly greater than that of nonmycorrhizal seedlings or seedlings inoculated with R. sindlis. Mycorrhizal stimulation of seedling growth was inhibited by nematode infection. When seedlings were inoculated with G. etunicatus arid R. similis, suppression of seedling growth by R. similis was less on VAM seedlings than on nonmycorrhizal seedlings, Nonmycorrhizal seedlings infected with R. similis were significantly smaller than nonmycorrhizal seedlings free of R. similis. Vesicle formation and mycelia growth were less in nematode-infected roots.     

Apoptosis induced by the flavonoid from lemon fruit (Citrus limon BURM. f.) and its metabolites in HL-60 cells

The flavonoid from lemon fruit (Citrus limon BURM. f.) and its metabolites, particularly eriodictyol, 3,4-dihydroxyhydrocinnamic acid, and phloroglucinol had the function of DNA fragmentation in HL-60 cells when analyzed by flow cytometry. An apoptotic DNA ladder and chromatin condensation were observed in HL-60 cells when treated with these compounds. The caspase inhibitor prevented DNA fragmentation. These compounds are anticipated to be useful for medical purposes.     

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Accumulation of Scoparone in Heat-Treated Lemon Fruit Inoculated with Penicillium digitatum Sacc

Phytoalexin scoparone (6,7-dimethoxycoumarin) generally was not detected in noninoculated lemon fruit (Citrus limon [L.] Burm., cv Eureka) but accumulated in fruit after inoculation with Penicillium digitatum Sacc. A much greater increase in the amount of scoparone was found in fruit exhibiting an incompatible response to Penicillium after heat treatment at 36°C for 3 days. Heat treatment prevented development of decay in the inoculated fruit. The concentration of the compound after inoculation continued to increase during and after the heat treatment period, reaching 178 micrograms per gram fresh weight of the flavedo 6 days after the heat treatment. Changes in scoparone concentration in fruit were closely correlated with the changes in the antifungal activity of the fruit extract. A low concentration of the phytoalexin was detected in fruit injured mechanically. Scoparone also accumulated in the fruit following ultraviolet illumination; the concentration of the compound was dose-dependent. Median effective dose values of the inhibition of germ tube elongation and spore germination of P. digitatum were 29 and 46 micrograms per milliliter, respectively. Our findings suggest that the rapid increase in scoparone concentration plays an important role in the increased resistance of heat-treated lemon fruit to infection by P. digitatum.     

Geraniol synthase whose mRNA is induced by host-selective ACT-toxin in the ACT-toxin-insensitive rough lemon (Citrus jambhiri)

Host-selective toxins (HSTs) produced by some strains of Alternaria alternata are selectively toxic to certain cultivars of plants. However, the role of HSTs in toxin-insensitive plants is currently unknown. Here, we studied the role of ACT-toxin using an ACT-toxin producing A. alternata strain SH20 and the ACT-toxin-insensitive plant rough lemon. Induction of some defense related genes in response to SH20 were faster or stronger than in response to the ACT-toxin deficient SH20 mutant. By sequencing subtractive PCR clones obtained from mRNA of rough lemon leaves inoculated with SH20 after subtraction with that of the ACT-toxin deficient SH20 mutant, we isolated the SH20-responsive genes in rough lemon. Among the SH20-responsive genes analyzed in this study, we isolated a terpene synthase (TPS) gene, RlemTPS3. We also determined that RlemTPS3 localizes to the chloroplast and produces the monoterpene geraniol.     

Growth responses and vascular plugging of citrus inoculated with rhizobacteria and xylem-resident bacteria

Summary Bacteria, isolated from roots (xylem tissue) of healthy and Young Tree Decline (YTD, Blight)-affected citrus trees, and also from nursery seedlings, were screened for potential pathogenicity by the tobacco hypersensitive reaction (HR). A majority (>75%) of the HR positive strains were classified as nonfluorescent pseudomonads. These HR positive strains were subsequently inoculated into rough lemon (Citrus jambhiri Lush.) and sweet orange (C. sinsensis Osbeck) seedlings or into Valencia sweet orange budded on rough lemon root-stock. Many of the HR positive pseudomonads reduced fresh weights (up to 94%) of roots and shoots and some reduced xylem water conductance and caused scion dieback. There was no evidence of necrosis or root rot in inoculated roots. A few HR negative Pseudomonas and Enterobacter strains significantly, but less severely, inhibited (to 43%) root growth of sweet orange seedlings. HR negative mutants derived from HR positive strains were considerably less inhibitory. Postinoculation stresses (dark and cold) markedly decreased susceptibility of seedlings to bacterial-induced inhibition. Evidence of cultivar-specific effects was obtained in comparable inoculations of rough lemon and sweet orange seedlings. Soil application of a fluorescent pseudomonad, which alone was growth stimulatory, intensified inhibitory effects of nonfluorescent, growth inhibitory, psuedomonads. This study demonstrates that many rhizobacteria isolated from xylem tissue of roots have detrimental effects on citrus.     

丛枝菌根真菌种间差异对柚苗营养生长及矿质含量的影响

在温室盆栽条件下研究了3种AM真菌Gigaspora margarita、Glomus mosseae和Glomus versiforme对长寿沙田柚(Citrus grandis cv. Changshou Shatian You)无菌苗营养生长及矿质含量的影响.结果表明,接种AM真菌的植株均有效地被感染;与对照相比,接种AM真菌能显著促进植株地上和地下部分生长,尤其促进了须根的生长,接种Glomus mosseae处理的主根长度比对照增加了22.7％,侧根数量增加了35.7％,须根数量和总长分别增加了160.8％和103.2％;接种AM真菌显著地提高了叶片的N、P、K、Ca、Mg、Zn、Cu和Mn含量,与对照相比,3种真菌处理的P含量分别增加了46.8％、88.7％和32.3％.3种AM真菌处理中,以接种Glomus mosseae处理营养生长最好,菌根依赖性最大,矿质元素N、P、K、Ca、Zn和Cu的含量最高,其效应顺序为Glomus mosseae＞Gigaspora margarita＞Glomus versiforme,可见,3种AM真菌对长寿沙田柚生长均有正效应,以Glomus mosseae最为显著,为长寿沙田柚适宜的优良菌种（株）.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

一氧化氮供体硝普钠浸种缓解盐胁迫对小麦种子萌发的抑制作用

以小麦品种‘德抗961＇为材料,用NO供体硝普钠（SNP）浸种研究外源NO对盐胁迫下小麦种子萌发的影响.结果表明：0.06 mmol/L的SNP浸种24 h后对盐胁迫下小麦种子发芽率、发芽指数、活力指数和吸胀速率的下调都有显著缓解作用;SNP浸种对盐胁迫下α-淀粉酶的活性无明显影响,但能显著提高盐胁迫下β-淀粉酶的活性;进一步研究表明,SNP浸种预处理对盐胁迫下的α-淀粉酶同工酶变浅的条带有所恢复（尤其是条带3）,同时使盐胁迫下变浅的β-淀粉酶同工酶的条带有明显的恢复（尤其是d、e、f、g）.并且SNP能显著降低盐胁迫下小麦地上部分和根中的Na^＋含量,提高其K＋含量,从而使K^＋/Na^＋显著提高.以上结果表明：SNP浸种预处理提高盐胁迫下小麦种子的萌发,主要是通过提高β-淀粉酶的活性来实现的.     

Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, and their mixtures

Antioxidant properties of individual essential oils from lemon (Citrus limon L.), pink grapefruit (Citrus paradise L.), coriander (Coriandrum sativum L.), and clove (Caryophyllus aromaticus L.) buds and their mixtures were studied by capillary gas-liquid chromatography. Antioxidant activity was assessed by oxidation of the aliphatic aldehyde hexanal to the carboxylic acid. The lowest and highest antioxidant activities were exhibited by grapefruit and clove bud essential oils, respectively. Mixtures containing clove bud essential oil also strongly inhibited oxidation of hexanal. Changes in the composition of essential oils and their mixtures in the course of long-term storage in the light were studied. The stability of components of lemon and coriander essential oils in mixtures increased compared to individual essential oils.     

Accumulation of HDMBOA-Glc is induced by biotic stresses prior to the release of MBOA in maize leaves

The effects of biotic stresses on the contents of benzoxazinones (Bxs) were investigated in maize leaves. When the causal agent of southern corn leaf blight, Bipolaris maydis, was inoculated on the third leaf, the amount of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) increased, reaching a maximum level 48 h after inoculation. The inoculation of weakly pathogenic Curvularia lunata and non-pathogenic Alternaria alternata also resulted in accumulation of HDMBOA-Glc, and filtrates of the cultures of B. maydis, C. lunata and A. alternata also showed the accumulation of elicitor-active compounds by the fungi. Furthermore the infection of B. maydis induced formation of dark brown lesions, where most abundant Bx-related compound was 6-methoxy-2-benzoxazolinone (MBOA). The later is formed by degradation of DIMBOA and HDMBOA, whereas HDMBOA-Glc was most abundant in the surrounding green tissues. Among the Bx-related compounds, MBOA exhibited the strongest inhibition of the germination of the conidia and of the growth of germ tubes of B. maydis, C. lunata and A. alternata. In addition to fungal infection, the feeding by rice armyworm larvae resulted in HDMBOA-Glc accumulation. These findings are discussed in relation to the possible ecological relevance of the conversion of DIMBOA-Glc into HDMBOA-Glc.     

Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark-beetle attack in Norway spruce (Pinaceae)

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.     

The plant growth-promoting fungus Aspergillus ustus promotes growth and induces resistance against different lifestyle pathogens in Arabidopsis thaliana

To deal with pathogens, plants have evolved sophisticated mechanisms including constitutive and induced defense mechanisms. Phytohormones play important roles in plant growth and development, as well as in the systemic response induced by beneficial and pathogen microorganisms. In this work, we identified an Aspergillus ustus isolate that promotes growth and induces developmental changes in Solanum tuberosum and Arabidopsis thaliana. A. ustus inoculation on A. thaliana and S. tuberosum roots induced an increase in shoot and root growth, and lateral root and root hair numbers. Assays performed on Arabidopsis lines to measure reporter gene expression of auxin-induced/ repressed or cell cycle controlled genes (DR5 and CycB1, respectively) showed enhanced GUS activity, when compared with mock-inoculated seedlings. To determine the contribution of phytohormone signaling pathways in the effect elicited by A. ustus, we evaluated the response of a collection of hormone mutants of Arabidopsis defective in auxin, ethylene, cytokinin, or abscisic acid signaling to the inoculation with this fungus. All mutant lines inoculated with A. ustus showed increased biomass production, suggesting that these genes are not required to respond to this fungus. Moreover, we demonstrated that A. ustus synthesizes auxins and gibberellins in liquid cultures. In addition, A. ustus induced systemic resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae DC3000, probably through the induction of the expression of salicylic acid, jasmonic acid/ethylene, and camalexin defense-related genes in Arabidopsis.     

Effects of Volatiles Evolved from Excised Plant Tissues and Lemon Oil on the Growth of Mung Bean Seedlings

Volatiles evolved from excised segments of leaves of oleander(Nerium oleander) and cedar (Cedrus deodara), peels of lemon(Citrus limon), orange (Citrus sinensis) and lime (Citrus aurantifolia),and roots of wasabi (Wasabia japonica) inhibited growth of mungbean seedlings. The volatiles evolved from lemon oil also markedlyinhibited elongation of mung bean seedlings. The active constituent(s)in the volatiles were distinct from ethylene. The mode of inhibitionby the volatiles from lemon oil was different from that of ethylene.The volatiles from lemon oil alleviated the inhibitory effectof ethylene on the growth of seedlings. The volatiles from lemonoil supressed the formation of lamellar structure in chloroplasts,thus resulting in etiolation of seedlings. (Received October 25, 1982; Accepted December 29, 1982)