Diterpene Phytoalexins Are Biosynthesized in and Exuded from the Roots of Rice Seedlings

Rice (Oryza sativa L.) produces a variety of diterpene phytoalexins, such as momilactones, phytocassanes, and oryzalexins. Momilactone B was previously identified as an allelopathic substance exuded from the roots of rice. We identified in this present study momilactone A and phytocassanes A–E in extracts of, and exudates from, the roots of rice seedlings. The concentration of each compound was of the same order of magnitude as that of momilactone B. Expression analyses of the diterpene cyclase genes responsible for the biosynthesis of momilactones and phytocassanes suggest that these phytoalexins found in roots are primarily biosynthesized in those roots. None of phytocassanes B–E exhibited allelopathic activity against dicot seedling growth, whereas momilactone A showed much weaker allelopathic activity than momilactone B. The exudation of diterpene phytoalexins from the roots might be part of a system for defense against root-infecting pathogens.     

Influence of Rhizoctonia solani on Egg Hatching and Infectivity of Rotylenchulus reniformis

The effects of culture filtrates of Rhizoctonia solani and root exudates of R. solani-infected cotton (Gossypium hirsutum) seedlings on hatching of eggs and infectivity of females of Rotylenchulus reniformis were evaluated in an attempt to account for the enhanced nematode reproduction observed in the presence of this fungus. Crude filtrates of R. solani cultures growing over sterile, deionized distilled water did not affect egg hatching. Exudates from roots of cotton seedlings increased hatching of R. reniformis eggs over that observed in water controls. Exudates from cotton seedling roots not infected or infected with R. solani did not differ in their effect on egg hatching. However, infection of cotton seedlings by reniform females was increased in the presence of R. solani, resulting in the augmented egg production and juvenile population densities in soil observed in greenhouse studies.     

Two new secondary metabolites isolated from Avena sativa L. (Oat) seedlings and their effects on osteoblast differentiation

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3–10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

Biosynthesis of pterocarpan and isoflavan phytoalexins in Medicago sativa: the biochemical interconversion of pterocarpans and 2′-hydroxyisoflavans

Feeding experiments have shown that 2′-7-dihydroxy-4′-methoxy-isoflavone-[Me-¹⁴C] and -isoflavanone-[Me-¹⁴C] are efficient precursors of the phytoalexins demethylhomopterocarpin, sativan and vesitol in CuCl₂-treated lucerne (Medicago sativa) seedlings. Demethylhomopterocarpin-[Me-¹⁴C] was also incorporated into sativan and vestitol, and vestitol-[Me-¹⁴C] was incorporated into demethylhomopterocarpin and sativan. Thus, the pterocarpan demethylhomopterocarpin and the 2′-hydroxy-isoflavan vestitol are interconvertible in M. sativa, but incorporation data, and the results of kinetic feeding experiments with l-phenylalanine-[U-¹⁴C] suggest that these compounds are synthesized simultaneously from a common intermediate, which could be involved in the interconversion. A carbonium ion, derived from an isoflavanol, a likely intermediate in the biosynthetic reductive sequence from 2′,7-dihydroxy-4′-methoxy-isoflavone and -isoflavanone, is proposed as this common intermediate. 7-Hydroxy-2′,4′-dimethoxyisoflavone-[4′-Me-¹⁴C] was a very poor precursor of all three phytoalexins. Sativan, then, is most probably derived by methylation of vestitol. The incorporation of vestitol-[Me-¹⁴C] into demethylhomopterocarpin, but not into maackiain, pterocarpan phytoalexins of red clover (Trifolium pratense), is also demonstrated.     

Effects of Capsaicin on Plant Growth

Rice (Oryza sativa L.) seedlings inhibited the growth of hypocotyls and roots of cress (Lepidium sativum L.) seedlings when both seedlings were grown together. Two growth inhibiting substances were found in the culture solution in which rice seedlings were hydroponically grown for 14 d. One growth inhibitor was further purified. This suggests that the rice seedlings may produce growth inhibiting substances, acting as allelochemicals to other plants, and release them from their roots into the environment.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

Genetic Structure and Aggressiveness of Rhizoctonia solani AG1‐IA,the Cause of Sheath Blight of Rice in Southern China

One hundred and eighty isolates of Rhizoctonia solani AG1‐IA, the causal agent of rice sheath blight, were obtained from six locations in southern China. The genetic structure of R. solani isolates was investigated using random amplified polymorphic DNA (RAPD) markers, and a considerable genetic variation among R. solani isolates was observed. Most of the genetic diversity was distributed within populations, rather than among them. The distribution pattern of the genetic variation of R. solani appears to be the result of high gene flow (Nm) and low‐genetic differentiation among populations. The aggressiveness of R. solani was visually assessed by rice seedlings of five different cultivars in the glasshouse. All isolates tested were found to induce significantly different levels of disease severity, reflecting considerable variation in aggressiveness. The isolates were divided into highly virulent, moderately virulent and weakly virulent groups, and the moderately virulent isolates were dominant in R. solani population. No significant correlation was observed among the genetic similarity, pathogenic aggressiveness and geographical origins of the isolates. Information obtained from this study may be useful for breeding for improved resistance to sheath blight.     

Effect of mycorrhization on the accumulation of rishitin and solavetivone in potato plantlets challenged with<Emphasis Type="Italic"> Rhizoctonia solani</Emphasis>

The effect of colonization with the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on the content of rishitin and solavetivone was determined in potato plants cv. Goldrush challenged with Rhizoctonia solani. Mycorrhization stimulated significantly the accumulation of both phytoalexins in roots of plantlets challenged with R. solani but did not influence phytoalexin levels in non-challenged plantlet roots. No accumulation of solavetivone or rishitin was detected in shoots. In Petri dish bioassays, rishitin and solavetivone inhibited mycelial growth of R. solani.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Suppression of Alfalfa Growth by Concommitant Populations of Pratylenchus penetrans and two Fusarium species

Growth of alfalfa (Medicago sativa cv. Vernal) seedlings was compared after inoculation with combinations of either Pratylenchus penetrans and Fusarium soloni or P. penetrans and F. oxysporum f. sp. medicaginis. A synergistic disease interaction occurred in alfalfa when F. oxysporum and P. penetrans were added simultaneously to the soil. Alfalfa growth was suppressed at all inoculum levels of P. penetrans and F. oxysporum, but not with F. solani. Seedlings inoculated with the nematode alone gave lower yields than when inoculated with either Fusarium species alone. Fusarium oxysporum, but not F. solani, was pathogenic to alfalfa under similar experimental conditions. Fusarium oxysporum did not alter the populations of P. penetrans in alfalfa roots, whereas the presence of F. solani was associated with a diminished number of P. penetrans in the roots.     

Screen of safflower (Carthamus tinctorius L.) genotypes against Phytophthora drechsleri and Fusarium solani,the causal agents of root rot disease

Safflower (Carthamus tinctorius L.) plants were affected by a severe root rot disease caused by Phytophthora drechsleri and Fusarium solani in Isfahan province of Iran during 2005–2007. Disease incidence was more than 30% in severely infected fields. Twenty-one safflower genotypes, including six local cultivars and 15 internal pure lines were evaluated for their resistance to root rot disease in laboratory and greenhouse conditions. Safflower seedlings were evaluated for lesion length on infected roots in laboratory, as well percentage of live seedlings in greenhouse. The results indicated a high negative correlation between lesion length on roots and percentage of live seedlings. The most resistant and susceptible genotypes to P. drechsleri were identified as pure line Karaj row 12 (KW12) and cultivar Koseh with lesion lengths of 10.01 and 15.51?mm on roots and 45.60 and 18.00% live seedlings, respectively. The most resistant genotype to F. solani was identified as pure line KW11 with a lesion length of 9.31?mm on roots as well 62.80% live seedlings. The most susceptible genotypes were identified as cultivar Koseh and pure lines KW2 and KW3 with lesion lengths of 13.29, 12.72 and 12.13?mm on roots and 25.60, 28.40 and 28.40% live seedlings, respectively. The most resistant genotypes to both P. drechsleri and F. solani were identified as pure lines KW15 and KW9 with a 55.40% live seedlings. The most susceptible genotypes were cultivars Koseh, Goldasht and pure lines KW6, KW3 and KW2 with 35.40, 35.40, 35.40, 37.60 and 37.60% live seedlings in greenhouse, respectively.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Effect of Age of Alfalfa Root on Penetration by Pratylenchus penetrans

Penetration by all migratory life stages of Pratylenchus penetrans into roots of alfalfa (Medicago sativa L. cv. Du Puits) was inversely proportional to tissue age. Two-day-old tissue in the root hair zone was penetrated twice as much as 10- or 20-day-old sections of the tap root. Age-related differences were also observed in branch roots; these differences were not affected by increasing the number of nematodes from 1 to 10 per inoculation site, nor by increasing the length of the incubation period from 6 to 96 h. Age-related differences were only significant with 3-wk-old plants, not with 2- and 1-wk-old seedlings. Nematodes entered roots at temperatures from 5 to 30 C with maximum entry at 20 C and minimum at 5 C. At all temperatures, except 5 C, penetration into young tissue (2 days) was significantly greater than into medium (10 days) and old (20 days) tissue. Females and third-stage larvae entered the different-aged root sections 122% and 83%, respectively, more than did males. Two-day-old seedlings of the alfalfa cultivars Vernal, Saranac, and Du Puits were penetrated equally by P. penetrans. Perhaps the inverse relationship between penetration and age of root is, in part, responsible for the increasing resistance or tolerance of plants to nematode damage as they grow older.     

Fungi and Gram-negative bacteria as soilborne minor pathogens of goat's rue (Galega orientalis Lam.)

Fungi and Gram-negative bacteria were isolated from inside the roots of field-grown goat's rue (Galega orientalis). Fungi were isolated from three plants out of a total of 45 tested. Two multinuclear Rhizoctonia solani isolates were identified to the anastomosis group 5 (R. solani AG-5-Gal) using pairings with known AG test cultures. One fungal isolate was identified to Phoma chrysanthemicola. Gram-negative bacteria were isolated from three plants out of 25 tested. They were identified using classical methods, the BIOLOG identification system based on the utilisation of 95 different carbon sources, and the MIDI system for the analysis of whole cell fatty acids. The two latter systems were computer-associated and utilised an extensive reference library of isolates. One bacterial isolate was identified as Enterobacter agglomerans and two isolates as Pseudomonas marginalis. R. solani AG-5-Gal reduced the emergence of Lupinus luteus, L. polyphyllus and french bean (Phaseolus vulgaris) and the growth of broad bean (Viciafaba), L. luteus and french bean, but did not cause obvious damage in goat's rue and pea (Pisum sativum). However, R. solani AG-5-Gal was re-isolated from the roots of all the test plant species following inoculation. P. chrysanthemicola reduced the emergence of L. polyphyllus and the growth of goat's rue, french bean and broad bean, and it was re-isolated from all of the test plant species (except for french bean) following inoculation. All the bacteria reduced the emergence of french bean, but not that of goat's rue and pea, when applied to the soil. When the roots were dipped into bacterial suspension, all the bacteria damaged french bean and L. polyphyllus. Additionally, P. marginalis JV3 damaged goat's rue and red clover. The pathogenicity of the fungi and bacteria were not changed when they were double-inoculated in pairs, except for R. solani AG-5-Gal and P. marginalis JV2 which reduced the emergence of goat's rue when inoculated together but not when inoculated separately.     

The Rhizoctonia-Meloidogyne Disease Complex in Flue-cured Tobacco

If Meloidogyne incognita preceded Rhizoctonia solani by 10 days or 21 days in roots of greenhouse-grown tobacco plants, root rot was more extensive than when the nematode and fungus were introduced either simultaneously or separately or when R. solani was added after artificial wounding. Histological examination of galled roots 72 days after inoculation with R. solani revealed extensive fungal colonization in the root-knot susceptible cultivar ''Dixie Bright 101'' when M. incognita preceded R. solani by 21 days. R. solani, normally nonpathogenic on mature tobacco roots, may cause severe losses when present with well-established root-knot nematode infections.     

Increase of chalcone synthase mRNA in pathogen-inoculated soybeans with race-specific resistance is different in leaves and roots

Soybeans (Glycine max [L.] Merr.) respond to pathogens by producing isoflavonoid-derived phytoalexins. Chalcone synthase (CHS) is the first enzyme of the flavonoid/isoflavonoid biosynthetic pathway. We investigated changes in the steady state levels of CHS mRNA and other specific mRNAs at increasing times after inoculation in two different race-specific interactions, one between leaves and the bacterium Pseudomonas syringae pv glycinea (Psg), and one between roots and the fungus, Phytophthora megasperma f. sp. glycinea (Pmg). The amount of CHS mRNA increases significantly in soybean leaves inoculated with an avirulent race of Psg but not with a virulent race or water. In contrast, the increase in CHS mRNA is similar in roots inoculated with zoospores of either an avirulent or virulent race of Pmg. CHS mRNA increases significantly in pathogen inoculated roots but not in uninoculated controls. Hydroxyproline-rich glycoprotein (HRGP) has been observed by others to increase in wounded or pathogen-inoculated plants. We report here that HRGP mRNA levels are greater in roots inoculated with an avirulent Pmg race than with a virulent race, but inoculation with either race causes a significant increase in HRGP mRNA with respect to controls. Calmodulin or ubiquitin mRNA do not increase in either uninoculated or inoculated roots and leaves. The possibility that race-specific resistance in soybeans is expressed differently in different organs of the plant is discussed.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Identification of a stress-induced protein in stem exudates of soybean seedlings root-infected with Fusarium solani f. sp. glycines

Sudden death syndrome of soybean (Glycine max) is caused by the soilborne fungus, Fusarium solani f. sp. glycines, that infects soybean roots. Besides root necrosis, symptoms include interveinal leaf chlorosis, necrosis and premature defoliation. It is proposed that a fungal toxin is produced in soybean roots and translocated to foliage. In this study, we isolated compounds from soybean stem exudates from plants that were either inoculated or not inoculated with F. solani f. sp. glycines. A protein with an estimated molecular mass of 17 kDa and designated as FISP 17 for F. solani f. sp. glycines-induced stress protein was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein occurred only in F. solani f. sp. glycines-infected soybean stem exudates. The N-terminal amino acid sequence of the purified protein had 100 % identity with a starvation-associated message 22 protein, and 80 and 78 % identity with purified bean pathogenesis-related proteins, PvPR1 and PvPR2, respectively. To determine if the protein was of plant or fungal origin, a synthetic peptide was designed based on the N-terminal sequence and used to raise a polyclonal antibody from rabbit. Western blot analysis showed that the antibody only reacted with a 17-kDa protein in F. solani f. sp. glycines-infected plant exudates, but no reaction occurred with healthy plant exudates or with culture filtrates of F. solani f. sp. glycines. This is the first report of the presence of a stress-induced protein in stem exudates of soybean seedlings root-infected with F. solani f. sp. glycines.