Interrelationships between Ethylene Production,Gall Formation,and Root-knot Nematode Development in Tomato Plants Infected with Meloidogyne javanica

Ethylene production was determined in excised tomato (Lycopersicon esculentum) root cultures of Meloidogyne javanica susceptible and resistant cultivars infected with M. javanica. Uninfected cultivars produced very low amounts of ethylene. Relatively high amounts of ethylene were produced by the infected susceptible cultivars. Peak production of 1.6 n moles * g root⁻¹ * h¹⁻ occurred between 9 and 16 days after inoculation (DAI). The period of high ethylene production coincided with that of rapid increase in gall weight. Low amounts of ethylene were also released by the infected resistant cultivar between 9 and 12 DAI, which follows the hypersensitivity reaction. Ethylene production in infected intact plants during the period of rapid gall growth was twice as much as in uninfected plants during the same time. Exposing excised root cultures to 0.5 or l0 ppm ethylene accelerated the rate of increase in gall weight of M. javanica infected roots. In contrast, overall root growth was inhibited by these treatments, compared to infected roots which were not exposed to ethylene.     

油茶叶肿病子房瘿体和叶片瘿体的形态结构特征观察

采用光学显微镜观察了油茶叶肿病子房瘿体和叶片瘿体的形态特征和组织结构特点。结果表明:(1)油茶叶肿病瘿体是油茶的幼嫩子房和幼叶被细丽外担菌感染后所导致的组织增生而成的,细丽外担菌分布于成熟子房瘿体的外表面和叶片瘿体的表面。(2)子房瘿体由受感染的雌蕊子房增殖而来,成熟后表皮层脱落。(3)子房壁分两层,外层子房壁肉质肥厚,主要由大型薄壁细胞构成,是食用的主要部分,内层子房壁为较薄的心皮层,内外两层子房壁之间具有较大的空隙;心皮内有胚珠,胚珠着生在中轴胎座上,胚珠内部中空而没有育性。(4)叶片瘿体为受感染的幼嫩叶片增殖所致,由大型薄壁细胞构成,瘿体表面具表皮层,成熟后表皮脱落。(5)在正常叶片与叶片瘿体的过渡区内有维管束相通,瘿体侧的厚度比正常叶片厚度增加3～4倍,细胞体积增大5～10倍,但瘿体细胞内无叶绿体。     

Effect of Inhibitors and Stimulators of Ethylene Production on Gall Development in Meloidogyne javanica-Infected Tomato Roots

Excised tomato roots infected with Meloidogyne javanica produced ethylene at 3-6 times the rate of noninfected roots. This increase in ethylene production started 5 days after inoculation. Gall growth and ethylene production in infected roots were accelerated by 1-aminocyclopropane-1-carboxylic acid (ACC), indole acetic acid (IAA), and ethrel known as ethylene production stimulators. When inhibitors of ethylene production, like aminoethoxyvinylglycine (AVG) or aminoxyacetic acid (AOA), or inhibitors of ethylene action like silver thiosulfate (STS), were applied, gall growth and ethylene production were inhibited. Enhanced expansion of parenchymatous cells was observed in sections from nematode-induced galls and ethylene-treated roots. Lignification of xylem elements and fibers in the vascular cylinder was markedly inhibited in the gall, compared with noninfected root tissue. Because ethylene is known to induce cell expansion and to inhibit lignification, it is suggested that this plant hormone plays a major role in the development of M. javanica-induced galls. Ethylene affects gall size by enhancing parenchymatous tissue development and allows expansion of giant cells and the nematode body by reducing tissue lignification.     

Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots

RNAi constructs targeted to four different genes were examined to determine their efficacy to reduce galls formed by Meloidogyne incognita in soybean roots. These genes have high similarity with essential soybean cyst nematode (Heterodera glycines) and Caenorhabditis elegans genes. Transformed roots were challenged with M. incognita. Two constructs, targeted to genes encoding tyrosine phosphatase (TP) and mitochondrial stress-70 protein precursor (MSP), respectively, strongly interfered with M. incognita gall formation. The number of galls formed on roots transformed with constructs targeting the M. incognita TP and MSP genes was reduced by 92% and 94.7%, respectively. The diameter of M. incognita inside these transformed roots was 5.4 and 6.5 times less than the diameter of M. incognita found inside control plants transformed with the empty vector. These results indicate that silencing the genes encoding TP and MSP can greatly decrease gall formation and shows a promising solution for broadening resistance of plants against this plant-parasitic nematode.     

ANATOMY OF SOME LEAF GALLS OF ROSA WOODSII (ROSACEAE)

Infection of Rosa woodsii by some members of the order Hymenoptera results in neoplasmic outgrowths on the leaves. One type of outgrowth produces a spherical swelling (leaf gall) while the other has extensive hair-like proliferations (hairy gall). The anatomy and ultrastructure of these galls were examined by light microscopy and transmission electron microscopy. The leaf gall cells were considerably larger than normal cells, lacked well-developed chloroplasts and were loosely arranged with prominent intercellular spaces. Vascular bundles were scattered throughout the gall tissue. The upper three cell layers of the leaf gall tissue resembles a periderm, having many suberin lamellae. The suberin lamellae were often traversed by pores which may represent incomplete plasmodesmata. Phenolic compounds were commonly seen both in the normal and gall cells. A layer of internal cells of the hairy galls have remarkably thickened cell walls, presumably due to the deposition of cellulosic substances. Unlike leaf galls, the epidermal cells of the hairy galls were not heavily cuticularized and no periderm was found. The hair-like outgrowths present on the outer surface of these galls had a central vascular bundle. The epidermis of the outgrowths also had thickened cell walls, and trichomes occurred on the outer surface. The structural modifications brought about by the insect invasion in these two galls are compared and their roles in gall formation are discussed.     

Crown-gall-resistant transgenic apple trees that Silence Agrobacterium tumefaciens oncogenes

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.these authors contributed equally to this workthese authors contributed equally to this work     

Transformation through agroinfection on decapitated shoot apex of field-growing <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A₄M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A₄M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A₄M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.     

Uromycladium tepperianum, the gall rust fungus from Falcataria moluccana in Malaysia and Indonesia

Batai (Falcataria moluccana) is a valuable tree species for forest plantations in Malaysia and Indonesia. Since 1993, a gall rust disease has caused severe damage to all growth stages, from seedlings in the nursery to mature trees in the field. To identify the fungus causing gall rust disease on F. moluccana in Malaysia and Indonesia, study of the mode of infection and changes in the anatomy of infected cells were carried out in the anatomy laboratory. The disease in Malaysia and Indonesia is caused by Uromycladium tepperianum. The fungus produces three longitudinally ridged teliospores on each head, with spores measuring 13–20 μm wide and 17–28 μm long. The fungus is microcyclic, completing its entire life cycle on F. moluccana. This study confirmed that the teliospores themselves cannot infect the host. Under favorable conditions, about 10 h after inoculation, teliospores germinate to produce basidiospores that form penetration pegs about 6 h later, and it is this peg which penetrates the host cells directly through the epidermis. Pycnia, recognized as small brown pustules, break through the epidermis about 7 days after inoculation.     

Root-knot development in mulberry infested with Meloidogyne incognita

Healthy mulberry roots were inoculated with second stage juveniles of the root-knot nematode, Meloidogyne incognita and the sequential anatomical changes in the root concurrent with the development of the nematode were studied with light and scanning electron microscopes. Visible anatomical changes occurred four weeks post-inoculation when the pear-shaped adult female nematodes appeared in the tissues. The infected roots lost their circular outline and increased in thickness as traumatic parenchyma was induced in the root. The stele was disorganised with reduced and deformed vascular elements. Many giant cells developed in the traumatic tissue, adjacent to the adult females. Eight weeks after the inoculation, cavities developed around the adult females by tissue disintegration, and the galls became discernable on the roots. The number and size of thegalls increased in the next three weeks, corresponding to the development of more traumatic tissues and the enlargement of the adult female nematodes. The majority of the nematodes were settled towards the gall periphery, with their posterior oriented outwards for the release of their eggs into soil by the disintegration of the outer root layers. But many were settled deep inside the gall tissue without a channel to release their eggs to soil. The inner cavities enlarged in large proportions, sometimes occupying the major volume of the gall. The conductive tissues of the root were totally disorganised or were nonexistent.     

The response of tomato seedling roots to infection by Verticillium albo-atrum or Fusarium oxysporum f. sp. lycopersici

The response of seedling roots of near-isogenic tomato varieties to infection by Verticillium albo-atrum or Fusarium oxysporum f. sp. lycopersici was investigated. Studies of the infection of seedling roots not artificially damaged indicated that there was an extra-vascular expression of resistance towards V. albo-atrum but not to F. oxysporum. Roots of resistant tomato seedlings infected by V. albo-atrum contained the fungus in the epidermis and outer cortex while susceptible roots became heavily colonised. Observations made by transmission electron microscopy showed that the fungus appeared to be abnormal in growth and appearance in the epidermal and cortical cells of resistant seedling roots but normal in susceptible roots. Two preformed antifungal terpenoids were detected in seedling roots in greater amounts in resistant that in susceptible varieties. The possible mechanisms of seedling root resistance to vascular wilts are discussed.     

Occurrence of Tetracampos ciliotheca (Cestoda: Bothriocephalidea) in the gall bladder of Clarias anguillaris in Burkina Faso

The presence of the bothriocephalidean cestode Tetracampos ciliotheca Wedl, 1861 is reported from the gall bladders of the catfish Clarias anguillaris from Loumbila and Ziga reservoirs, Burkina Faso, in 2010–2011. Of the 159 specimens examined, 47 were infected. Mean intensity of infection was 4.3 parasites per infected host, the maximum was 22 tapeworms in the gall bladder and the mean abundance was 1.3 parasites. There was no significant difference between males and females or between the two localities. There was a positive relationship between fish size and the number of parasites in the gall bladder. This study provides further evidence of the presence of T. ciliotheca in the gall bladder, in which most tapeworms found were localised. Tapeworms were also found in the stomach and intestine, with prevalences of 1% and 16%, and mean infection intensities of 1 and 5.3, respectively.     

Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

Anatomical studies of Ustilago galls on Hordeum vulgare L.

The present paper deals with the histopathological studies of galls of Ustilago hordei (Pers.) on Hordeum vulgare L. The gall formation in the present case is the result of both hyperplastic and hypertrophic activities of the infected host cells. Also the gall formation by U. hordei on. H. vulgare is being reported for the first time through present. communication.     

Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz

Trichosanthin (TCS) from Trichosanthes kirilowii Maximowicz (T. kirilowii) can be used to treat choriocarcinoma. In this work, we established a novel system to produce TCS in crown gall tissues of T. kirilowii infected by Agrobacterium tumefaciens C58 (A. tumefaciens). In the crown gall tissues, a nopaline synthase (NOS) gene of A. tumefaciens was identified by polymerase chain reaction (PCR), and nopaline accumulation was confirmed by a high-voltage filter paper electrophoresis. Furthermore, we optimized conditions to culture the crown gall tissues able to grow fast and produce TCS in an auxin-free medium, and found that a fungal elicitor of Armillaria mellea was capable of stimulation of TCS secretion into the medium. Moreover, we identified that the TCS purified from the crown gall tissues could induce gastric cancer cell death. These data underscore the usefulness of our system as an inexpensive and virtually unlimited source of TCS.     

Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato

A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode–plant interaction.     

DNA from Agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues

Summary Axenic root tissue cultures were established from primary hairy roots induced on carrot and potato by Agrobacterium rhizogenes strain 15834. cDNA made towards poly-A⁺ RNA isolated from these tissues, hybridized with a limited number of well-defined fragments of the plasmid DNA present in the inciting A. rhizogenes strain. These data therefore demonstrate that at least part of the rootinducing (Ri) plasmid of Agrobacterium rhizogenes is transferred, stably maintained and expressed in hairy-root plant tissues and confirm that hairy roots are a special type of crown gall. The T-DNA in hairy-root cells appears to have several regions which are related in terms of sequence homology and probably also function to the T-DNA in octopine and nopaline crown gall tumours.     

Metabolism and Plant Hormone Action During Clubroot Disease

Infection of Brassicaceae with the obligate biotrophic pathogen Plasmodiophora brassicae results in the development of root galls (clubroots). During the transformation of a healthy root to a root gall a plethora of changes in primary and secondary metabolism occur. The upper part of an infected plant is retarded in growth due to redirection of assimilates from the shoot to the root. In addition, changes in the levels of plant growth regulators, especially auxins and cytokinins, contribute to the hypertrophy of infected roots. Also, defense reactions are manipulated after inoculation of suitable host plants with P. brassicae. This review summarizes our current knowledge on the changes in these parameters. A model is presented for how primary metabolism and secondary metabolism, including plant hormones, interact to induce clubroot formation.     

Galling and reversion disease incidence in a range of blackcurrant genotypes, differing in resistance to the blackcurrant gall mite (Cecidophyopsis ribis) and blackcurrant reversion disease

An assessment was made of the response of 10 blackcurrant genotypes, differing in resistance to the agent of blackcurrant reversion disease (BRD) and to its gall mite vector, in field trials in Scotland and Finland over 5 yr. At each location, the 10 genotypes were planted in plots containing infector plants with high inoculum levels of the two organisms. In Scotland, the infector plants contained large numbers of gall mites (Cecidophyopsis ribis) and were infected with the European (E) form of BRD; in Finland, infector plants contained a different species of gall mite (C. spicata) and the severe Russian form (R) of BRD. As expected, at both sites, almost all plants of cvs Ben Alder, Ben Lomond, Ben Tirran, Ojebyn and an SCRI selection F4/1/66, which are susceptible to gall mite and BRD, became infested with each of these organisms. However, in Scotland but not in Finland, 58% of cv. Ojebyn plants were affected by BRD. The cv. Foxendown, which contains gene Ce that confers apparent immunity to C. ribis, was free from galls and failed to develop distinctive BRD symptoms at both sites. The cvs Rus and Neosypajuscajaija, which contain gene P which is reported to confer resistance to C. ribis, were infested more slowly by mites than the mite-susceptible genotypes and showed a smaller number of galls per plant. Also, they were infected with BRD more slowly than some mite-susceptible genotypes although by the end of the experiment, most plants were affected by BRD. All plants of cvs Golubka and Ben Gairn, which are resistant to the agent of BRD, remained free from distinct BRD symptoms at both sites despite the fact that plants contained galls. These data indicate the superiority of gene Ce over gene P for resistance to gall mites with the added benefit that the virtual immunity to gall mites provided by gene Ce provides a high level of protection against infection with BRD. The relative merits of these different forms of resistance to gall mites and BRD in blackcurrant are discussed in relation to present control methods for these two organisms and in the light of recent findings of different species of eriophyid mites on Ribes species.     

STRANGLER FIG ROOTING HABITS AND NUTRIENT RELATIONS IN THE LLANOS OF VENEZUELA

The strangler figs, Ficus pertusa and F. trigonata, are abundant in the seasonally flooded palm savanna (llanos intermedio) near Calabozo, Venezuela. The most common host tree for the hemiepiphytic figs is the palm Copernicia tectorum; nearly half of the palms support either an epiphytic or a ground-rooted fig. During their epiphytic stage the figs are rooted behind the palms' marcescent leaf bases. Material trapped behind the leaf bases is higher in organic matter, nitrogen, magnesium, and potassium than soil from the ground near the palms. The suggestion that nutrient availability to epiphytes is high is supported by the observation that concentrations of several nutrients, including N, P, and K, are significantly higher in epiphytic leaves than in tree leaves. Figs retain access to the epiphytic medium by producing upwardly growing (apogeotropic) roots that remain attached in the host palm's crown long after the fig has become firmly rooted in the ground. Although upward growing roots are expected to be more important in nutrient than water uptake, there are no obvious differences in the xylem anatomy of upward and downward growing fig roots. Terrestrial roots of fig trees are generally infected with vesicular-arbuscular mycorrhizae, but the epiphytic roots of the same individuals are not infected.