Reaction of soybean callus to culture filtrates of Phialophora gregata

Soybean [Glycine max (L.) Merr.] calli derived from susceptible and resistant soybean genotypes were exposed to the culture filtrates of pathogenic and non-pathogenic isolates of Phialophora gregata (Allington and Chamberlain) W. Gams. The rate of browning, growth and viability (measured by 2,3,5-triphenyltetrazolium chloride reduction) of the callus were determined after various exposure times to the fungus culture filtrates. Callus from susceptible Century, Cumberland, Corsoy 79, Harosoy and Clark 63 were sensitive to the culture filtrates of pathogenic isolates of P. gregata. Callus from Plant Introductions 437833 and 84946-2, when treated with fungal culture filtrates, did not develop browning and callus growth and cell viability were not decreased compared to untreated controls. Culture filtrates from non-pathogenic isolates of the fungus did not affect the growth of susceptible and resistant callus. Tobacco (Nicotiana tabacum L.) callus was not sensitive to the culture filtrate of a P. gregata isolate pathogenic to soybean. The fungal culture filtrate, based on limited evaluation, appears to be selective towards soybean callus. Based on this initial work, it appears that soybean callus bioassays have utility for evaluating soybean for resistance to P. gregata as well as assessing pathogenicity of fungus isolates.     

Evaluation of soybean resistance to Phialophora gregata culture filtrate in tissue culture

Summary Resistance to the fungal pathogen, Phialophora gregata (Allington and Chamberlain) W. Gams, the cause of brown stem rot (BSR) in soybean [Glycine max (L.) Merr.], is an important trait for cultivars grown in the northern USA. A novel tissue culture method was developed where ten soybean cultivars were differentiated on the ability of their excised cotyledons to remain green and initiate callus in a tissue culture medium containing P. gregata culture filtrate. Cultivar BSR classifications by the cotyledon method corresponded to greenhouse root-dip assay classifications in 80%, 100%, and 90% of the three P. gregata isolate treatments. Another method, employing pieces of somatic callus exposed to the culture filtrate, had a 70% average correspondence to the greenhouse results. Physiologic specialization was demonstrated in parallel in vivo/in vitro assays for the first time. These data suggest that the cotyledon method would accurately identify soybean lines resistant to certain aberrant or wild-type P. gregata isolates.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

Histopathology of Susceptible and Resistant Capsicum annuum Cultivars Infected with Ralstonia solanacearum

The vascular colonisation of resistant and susceptible hot chilli (Capsicum annuum) cultivars by Ralstonia solanacearum was examined using transmission electron microscopy. Tap roots of artificially-inoculated plants, grown in sterilised soil were investigated to observe the morphological barriers involved in the restriction of bacterial spread. In the resistant cultivar, several responses induced in response to bacterial infection, were observed. First, a cell wall coating material developed together with swelling of the primary wall of the xylem vessels, limiting the bacterial spread. Second, formation of various types of vesicles in the vascular parenchyma cells, which enveloped the bacterial mass and also partly restricted the pathogen spread. Third, induction of hypersensitive reaction in the xylem vessels resulted in the distortion and lysis of the bacteria. In the susceptible cultivar, vascular coating, production of vesicle and induction of hypersensitive reaction were not observed and bacterial spread was not limited. Rapid vascular colonisation of the susceptible cultivar seemed to be generalised which resulted in the rapid wilting of affected plants. Other reactions involved in both resistant and susceptible cultivars include disorganisation of cytoplasm of parenchyma cells, disintegration of nuclei, and rupturing of xylem vessel walls. The restriction of pathogen spread associated with the resistance in C. annuum to bacterial wilt was mainly attributed to some induced, morphological and physical barriers.     

Cytology of infection,development and expression of resistance to Plasmodiophora brassicae in canola

The timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi‐nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of non‐inoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.     

Effect of Within-field Variation in Soil Texture on Heterodera glycines and Soybean Yield

The influence of soil texture on Soybean yield in the presence of Heterodera glycines was investigated by comparing yields of susceptible cultivars with a resistant cultivar for 2 years. Soybean yield was negatively correlated with increasing sand content (P = 0.05). Yields of susceptible cultivars were suppressed with increasing sand content. Final nematode population densities were lowest in plots with greatest sand content. Soybean infection by SCN, as determined by the number of cysts 30 days after planting, was not consistently related to soil texture over 2 years. Initial nematode population density was positively related to soybean yield the first year and negatively related to soybean yield the second, probably a result of greater yield suppression by H. glycines in plots with greater sand content.     

Effect of Brown Stem Rot on Water Relations of Soybean

To investigate the effects of brown stem rot, a vascular disease of soybean (Glycine max) induced by Phialophora gregata, on the water relations of diseased plants, stems of greenhouse-grown plants of susceptible (Pride B216) and resistant (BSR 201) cultivars were injected with the pathogen at vegetative growth stage VI. Plants of both cultivars developed internal stem browning, but those of Pride B216 developed more severe symptoms of water stress (reduced leaf water potential and stem conductance). Inoculated plants of both cultivars also had reduced stem conductance and increased stomatal conductance and transpiration. Disease-related water stress can be attributed to the combined effects of reduced stem conductance and increased water loss resulting from increased stomatal conductance.     

Diversity of Rhizosphere Soil Arbuscular Mycorrhizal Fungi in Various Soybean Cultivars under Different Continuous Cropping Regimes

Recent studies have shown that continuous cropping in soybean causes substantial changes to the microbial community in rhizosphere soil. In this study, we investigated the effects of continuous cropping for various time periods on the diversity of rhizosphere soil arbuscular mycorrhizal (AM) fungi in various soybean cultivars at the branching stage. The soybean cultivars Heinong 37 (an intermediate cultivar), Heinong 44 (a high-fat cultivar) and Heinong 48 (a high-protein cultivar) were seeded in a field and continuously cropped for two or three years. We analyzed the diversity of rhizosphere soil AM fungi of these soybean plants at the branching stage using morphological and denaturing gradient gel electrophoresis (DGGE) techniques. The clustering analysis of unweighted pair-group method with arithmetic averages (UPGMA) was then used to investigate the AM fungal community shifts. The results showed that increasing the number of years of continuous cropping can improve the colonization rate of AM fungi in different soybean cultivars at the branching stage. The dominant AM fungi in the experimental fields were Funneliformismosseae and Glomus spp. The number of years of continuous cropping and the soybean cultivar both had obvious effects on the diversity of AM fungi, which was consistent with the results of colonization rate analysis. This study establishes a basis for screening dominant AM fungi of soybean. In addition, the results of this study may be useful for the development of AM fungal inoculants.     

Kinetics and Histopathology of the Cacao-Ceratocystis cacaofunesta Interaction

Theobroma cacao L., the chocolate tree, is an important tropical tree-crop that provides sustainable economic and environmental benefits to some of the poorest and most ecologically sensitive areas of the world. Ceratocystis wilt (CW), caused by the fungus Ceratocystis cacaofunesta, is a highly dangerous disease capable of killing the cacao plant. Histopathological studies of the host–pathogen interaction are an effective resource to study pathogenesis which will ultimately lead to a better elucidation of the mechanisms of host resistance. We analyzed the development of C. cacaofunesta reproductive structures, the pattern of fungal colonization and the anatomical responses of cacao resistant (TSH 1188) and susceptible (CCN 51) genotypes to CW. Results showed that conidia germination started within 4 h after inoculation (hai) in agar, by 12 hai conidia germination was 80 %. The resistant genotype showed little or no fungal colonization compared to the susceptible genotype. Host reactions were characterized by greater quantities of gels, gums and tyloses in the susceptible genotypes compared to the resistant. The susceptible genotype had a large number of cells surrounding the xylem vessels that were infused with dark brown phenolic compounds. The host defense reaction observed near the xylem in the resistant genotype, suggest that a mechanism involved with resistance to C. cacaofunesta.     

Association of the Production of Phenylpropanoid Compounds at the Infection Sites of Corynespora cassiicola with Soybean Resistance against Target Spot

This study investigated, at the microscopic level, whether the differential defence responses of soybean cultivars that are resistant (Fundacep 59) and susceptible (TMG 132) to target spot, caused by Corynespora cassiicola, could be associated with an increase in the production of phenolics, flavonoids and lignin at the infection sites. Many larger necrotic lesions with yellow halos were noticed on the leaves of plants from cultivar TMG 132, in contrast to the leaves of plants from cultivar Fundacep 59. Necrotic lesions also developed on the petioles of leaves of plants from cultivar TMG 132, while on the petioles and veins of leaves of plants from cultivar Fundacep 59, the lesions were of purple colour. The growth of fungal hyphae was reduced on the leaves of plants from cultivar Fundacep 59, and an apparently high density of trichomes was found in comparison with the leaves of plants from cultivar TMG 132. An appressorium‐like structure was produced at one or both extremities of the conidium of C. cassiicola, preferentially at the major and minor veins on the adaxial leaf surface of plants from both cultivars. Most cells on the leaves of plants from cultivar Fundacep 59 reacted against C. cassiicola infection by accumulating phenolic‐like compounds, which contributed to the death of many fungal hyphae and a greater maintenance of cell integrity. In contrast, fungal hyphae grew without any impedance in the leaf cells of plants from cultivar TMG 132, which was associated with signs of intense leaf tissue disorganization. Stronger autofluorescence and deposition of lignin and flavonoids were found in the cells of leaves of plants from cultivar Fundacep 59, in contrast to cultivar TMG 132. It can be concluded that soybean resistance to target spot is probably dependent on the activation of the phenylpropanoid pathway.     

Herbivore-induced indirect defense across bean cultivars is independent of their degree of direct resistance

We tested the extent to which resistance of common bean (Phaseolus vulgaris) cultivars to the spider mite Tetranychus urticae parallels the extent to which these plants display indirect defenses via the induced attraction of the predatory mite Phytoseiulus persimilis. First, via field and greenhouse trials on 19 commercial bean cultivars, we selected two spider mite-resistant (Naz and Ks41128) and two susceptible (Akthar and G11867) cultivars and measured the spider mite-induced volatiles and the subsequently induced attraction of predatory mites via olfactory choice assays. The two major volatiles, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl-acetate, were induced in the resistant but not in the susceptible cultivars. However, uninfested susceptible cultivars emitted these volatiles at levels similar to those of mite-infested resistant cultivars. Significant induction of several minor components was observed for all four cultivars except for the infested-susceptible cultivar G11867. Both, the spider mite-resistant cultivar Naz and the susceptible cultivar G11867, attracted more predatory mites when they were infested. In contrast, spider mites induced increased emission of two major and five minor volatiles in Ks41128, but predatory mites did not discriminate between infested and uninfested plants. Overall, the attraction of predatory mites appeared to correlate positively with the presence of TMTT and (Z)-3-hexenyl acetate and negatively with β-caryophyllene and α-pinene in the bean headspace. Taken together, our data suggest that resistance and attraction of natural enemies via induced volatiles are independent traits. We argue that it should be possible to cross predator-attraction promoting traits into resistant cultivars that lack sufficiently inducible indirect defenses.     

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

Background

Plant systemic signaling characterized by the long distance transport of molecules across plant organs involves the xylem and phloem conduits. Root-microbe interactions generate systemic signals that are transported to aerial organs via the xylem sap. We analyzed the xylem sap proteome of soybean seedlings in response to pathogenic and symbiotic interactions to identify systemic signaling proteins and other differentially expressed proteins.

Results

We observed the increase of a serine protease and peroxidase in the xylem sap in response to Phytophthora sojae elicitor treatment. The high molecular weight fraction of soybean xylem sap was found to promote the growth of Neurospora crassa in vitro at lower concentrations and inhibit growth at higher concentrations. Sap from soybean plants treated with a P. sojae elicitor had a significantly higher inhibitory effect than sap from control soybean plants. When soybean seedlings were inoculated with the symbiont Bradyrhizobium japonicum, the abundance of a xyloglucan transendoglycosyl transferase protein increased in the xylem sap. However, RNAi-mediated silencing of the corresponding gene did not significantly affect nodulation in soybean hairy root composite plants.

Conclusion

Our study identified a number of sap proteins from soybean that are differentially induced in response to B. japonicum and P. sojae elicitor treatments and a majority of them were secreted proteins.     

Impact of Herbicides on Heterodera glycines Susceptible and Resistant Soybean Cultivars

Several abiotic and biotic stresses can affect soybean in a growing season. Heterodera glycines, soybean cyst nematode, reduces yield of soybean more than any other pathogen in the United States. Field and greenhouse studies were conducted to determine whether preemergence and postemergence herbicides modified the reproduction of H. glycines, and to determine the effects of possible interactive stresses caused by herbicides and H. glycines on soybean growth and yield. Heterodera glycines reproduction factor (Rf) generally was less on resistant than susceptible cultivars, resulting in a yield advantage for resistant cultivars. The yield advantage of resistant cultivars was due to more pods per plant on resistant than susceptible cultivars. Pendimethalin reduced H. glycines Rf on the susceptible cultivars in 1998 at Champaign, Illinois, and in greenhouse studies reduced dry root weight of H. glycines-resistant and susceptible cultivars, therefore reducing Rf on the susceptible cultivars. The interactive stresses from acifluorfen or imazethapyr and H. glycines reduced the dry shoot weight of the resistant cultivar Jack in a greenhouse study. Herbicides did not affect resistant cultivars'' ability to suppress H. glycines Rf; therefore, growers planting resistant cultivars should make herbicide decisions based on weeds present and cultivar tolerance to the herbicide.     

Development of Meloidogyne arenaria on Peanut and Soybean under Two Temperature Cycles

Florunner peanut and three soybean cultivars, Centennial, Gasoy 17, and Wright, were inoculated with 48-hour age cohorts of Meloidogyne arenari race 1 second-stage juveniles and placed in a growth chamber set to simulate early season (low temperature) and midseason (high temperature) conditions. Percentages of the initial inoculum penetrating roots 4 and 8 days after inoculation were 2-3 times higher in soybean cultivars than in peanut; 25% on susceptible soybean and 9% on peanut. Penetration and early development of M. arenaria were greater in the higher temperature environment. Penetration percentages were expressed as a function of cumulative degree-days by regression models. Development of M. arenaria 10, 20, and 30 days after inoculation was more rapid on peanut than on soybean. The resistant soybean cultivar Wright had slower development rates than did the other two soybean cultivars. Nematode growth and development were dependent on temperature. In greenhouse experiments, production of eggs by M. arenaria was more than 10 times greater on peanut than on susceptible soybean. The reproductive factor for Wright soybean was less than one, but plant growth parameters indicated that this cultivar was intolerant of M. arenavia.     

Partitioning two- and three-trophic-level effects of resistant plants on the predator, Nabis roseipennis

Two- and three-trophic-level effects of resistant soybean plants on the development and fecundity of the predator Nabis roseipennis Reuter (Hemiptera: Nabidae) were determined. The nabids, like many hemipteran predators, feed on plants as well as arthropod prey. The life history traits of N. roseipennis when feeding on Pseudoplusia includens (Hübner) (Lepidoptera: Noctuidae) fed the resistant soybean cultivar PI 229358 or the susceptible Cobb' and while exposed to these resistant and susceptible soybean cultivars or a no-plant control were determined. Nymphal survival of N. roseipennis was unaffected by either prey or plant treatment. Development time was extended and fecundity of N. roseipennis was reduced by feeding on prey fed the resistant plant but not by exposure to resistant soybean plants. Thus, three-trophic-level effects of the resistant cultivar PI 229358 on prey quality were more important than two-trophic-level effects of direct plant feeding on development and fecundity of N. roseipennis. Consistency of these results with those of other predatory hemipterans and the potential impact of diet composition on the effects of resistant plants are discussed.     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Influence of plant resistance at the third trophic level: interactions between parasitoids and entomopathogenic fungi of cereal aphids

Host-plant resistance can affect herbivorous insects and their natural enemies such as parasitoids and entomopathogenic fungi. This tritrophic effect acts on interspecific interactions between the two groups of natural enemies distantly related in phylogenetic terms. The intra- and extra-host aspects of the interaction between the cereal aphid parasitoid Aphidius rhopalosiphi and the entomopathogenic fungus Erynia neoaphidis developing on the grain aphid, Sitobion avenae, on resistant and susceptible wheat (Triticum aestivum) cultivars, were studied. The competitive outcome of the intra-host interaction depended on the timing of parasitoid oviposition and fungal infection and was affected by wheat resistance. In particular, survival of the parasitoid was lower on the resistant wheat cultivar than the susceptible wheat cultivar, when the competitive outcome of the interaction was favourable for either parasitoid or fungal development. Before and after this period the influence of plant resistance was not significant. Furthermore, the extra-host interaction was not affected by the wheat cultivar, although an increase in fungal infection of S. avenae was observed when parasitoids foraged in the experimental arena with sporulating aphid cadavers compared with foraging in the absence of sporulating cadavers. Our results showed that the host plant may affect interspecific interactions between parasitoids and fungi and that these interactions depended on the timing of parasitoid oviposition and fungal infection. Received: 16 March 1998 / Accepted: 24 August 1998