A stilbene synthase gene (SbSTS1) is involved in host and nonhost defense responses in sorghum

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.     

Defense gene expression in Sorghum bicolor against Macrophomina phaseolina in leaves and roots of susceptible and resistant cultivars

The fungal disease, charcoal root rot, caused by Macrophomina phaseolina is a foremost yield restraining factor of Sorghum bicolor L. around the world. The expression analysis of genes induced in general defense response can endow with clues to reveal major defense mechanisms against pathogen infection in sorghum plant. The role of chitinase and Stilbene synthase in response to M. phaseolina in sorghum was studied under control growth conditions using a real-time polymerase chain reaction. Here, we report the expression analysis of antifungal genes in two cultivars viz. PJ-1430 (resistant) and SU-1080 (susceptible) at different hours after inoculation with M. phaseolina isolate MTCC 2165. Chitinase and stilbene synthase were induced in PJ-1430 within 0 h, 24 h and in SU-1080 in 48 h, 24 h, respectively, after inoculation. However, the expression levels of chitinase and stilbene synthase in resistant cultivar were significantly higher. The results showed that chitinase and stilbene synthase can be effective to enhance resistance to M. phaseolina.     

Constitutive accumulation of cis-piceid in transgenic Arabidopsis overexpressing a sorghum stilbene synthase gene

Sorghum SbSTS1 was the first example of a stilbene synthase gene in monocots. Previously, we demonstrated that the gene was involved in defense responses. To examine its biochemical function in planta, SbSTS1 was overexpressed in transgenic Arabidopsis. Metabolite analysis revealed that cis-resveratrol glucoside (piceid) accumulated as the major stilbene in the transgenic lines. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode, up to 580 microg g(-1) FW of cis-piceid were detected in 2-week-old plants, which represent a convenient source of the cis-isomers for pharmacological investigations. Our results also suggested the presence of unknown stilbene isomerase activities in Arabidopsis.     

Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry

In a previous report, it was described that strawberry plants pre-treated with an avirulent isolate of Colletotrichum fragariae (M23) acquired resistance to a virulent isolate of Colletotrichum acutatum (M11) causing anthracnose. In this report we present evidence that the eliciting activity can be found not only in conidial extracts but in culture supernatants of the avirulent pathogen as well. Plants of the cv. Pájaro treated with the culture filtrate (CF) derived from M23, 3 days prior to the inoculation with M11 showed significantly reduced disease severity as compared to control plants and the disease was completely suppressed when plants were pre-treated 7 days before the challenge inoculation with M11. The same effect was achieved when a single leaf was sprayed with CF, suggesting that the resistance acquired is systemic. Control treatments showed that none of the active extracts inhibited the growth of the virulent pathogen, indicating that the protection effect was due to the induction of a defense response. The latter was confirmed by the accumulation of reactive oxygen species (e.g. hydrogen peroxide, superoxide anion) and the deposition of lignin and callose, usually associated to plant defense, after the CF treatment. Experiments carried out with other strawberry cultivars treated with CF showed that also protected them against different virulent isolates, suggesting that the response observed is cultivar-nonspecific. These outcomes indicate that the protection against anthracnose in strawberry involves a phenomenon of induced resistance (IR) by action of defense-eliciting molecules produced by M23.     

Interrelationships Among Macrophomina phaseolina,Criconemella xenoplax,and Tylenchorhynchus annulatus on Grain Sorghum

Microplot experiments were established in 1992, 1993, and 1994 to investigate the relationships among Macrophomina phaseolina, Criconemella xenoplax, mad Tylenchorhynchus annulatus on grain sorghum in Louisiana. A factorial treatment arrangement of two grain sorghum hybrids (De Kalb DK 50 and Pioneer hybrid 8333), three levels of M. phaseolina (0, 10, and 100 colony-forming units (CFU)/g soil), and three nematode inoculum levels (0, 1x, and 2x) were used. Nematode inocula at 1x levels were 929, 1,139, and 1,445 C. xenoplax and T. annulatus/microplot in 1992, 1993, and 1994, respectively. Plants were harvested after 90-105 days. In all 3 years, grain sorghum root and head dry weights were suppressed as nematode inoculum level increased. These reductions were detected both in the absence and in the presence of M. phaseolina at 10 CFU/g. Reproduction of both nematode species was suppressed by M. phaseolina. Interactions between M. phaseolina and nematodes were antagonistic with regard to plant dry weights, yield, and nematode reproduction, so that combined effects were less than the sum of the effect of each pathogen alone.     

Histopathology of Pea Roots Axenically Infected by Pratylenchus penetrans

The histological changes in pea roots axenically infected by Pratylenchus penetrans were studied and described. Roots of pea seedlings growing aseptically on the surface of nutrient agar slants were inoculated with axenized nematodes. Six hours after inoculation most of the nematodes introduced were probing the root epidermis, but none had completely entered though a few were observed with their anterior section already in the root. Most of the nematodes penetrated the roots after 12 hr inoculation. From 18 to 24 hr after inoculation the nematodes were mostly in the mid-cortex. Invaded regions of the cortex often showed orange discoloration. As incubation continued, the number of nematodes in these roots increased, and feeding and reproductive activities extended deeper into the cortex. These activities resulted in extensive breakdown of the cortex. No nematodes were observed within the stele of infected roots; however, the endodermis of infected roots stained dark-brown. Gravid female nematodes probed the root endodermis and some endodermal cells appeared to collapse after prolonged probing by the nematode. All stages in the life cycle of the nematode were observed in infected roots; the female to male ratio inside the root was about 5:1.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.     

Genetical and biological control of cotton ashy stem caused by Macrophomina phaseolina in outdoor pot experiment

Two outdoor pot experiments were carried out to evaluate the reaction of 11 commercial Egyptian cotton cultivars Macrophomina phaseolina, the incitant of ashy stem in cotton and to evaluate the antagonistic ability of 27 isolates of Trichoderma sp. against pathogen cotton cultivars Giza 85, Giza 87, Giza 89 and Giza 90 were resistant to M. phaseolina because both survival and plant height of these cultivars was not affected when the soil was infested with the pathogen. None of the cultivars were found to be immune to highly pathogenic of M. phaseolina isolate. Of the 27 isolate’s of Trichoderma that were evaluated, the best antagonistic performance was given by isolates nos. 2, 10, and 16 were promising for commercialization because they significantly increased survival and improved plant height and dry weight of the surviving cotton seedlings.     

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.     

Population Dynamics of Plant-parasitic Nematodes on Cover Crops of Corn and Sorghum

Buildup of plant-parasitic nematode populations on corn (Zea mays), soybean (Glycine max), and sorghum (Sorghum bicolor) were compared in 1991 and 1992. Final population densities (Pf) of Meloidogyne incognita were lower following sorghum than after soybean in both seasons, and Pf after sorghum was lower than Pf after corn in 1992. In both seasons, Pf differed among the sorghum cultivars used. No differences in Pf on corn, sorghum, and soybean were observed for Criconemella spp. (a mixture of C. sphaerocephala and C. ornata) or Paratrichodorus minor in either season. Pf levels of Pratylenchus spp. (a mixture of P. brachyurus and P. scribneri) were greatest after corn in 1992, but no differences with crop treatments were observed in 1991. When data from field tests conducted with corn and sorghum during the past four seasons were pooled, negative linear relationships between ln(Pf/Pi) and ln(Pi) were observed for Criconemella spp. and P. minor on each crop, and for M. incognita on corn (Pi = initial population density). Although ln(Pf/Pi) and ln(Pi) were not related for M. incognita with pooled sorghum data, separate relationships were derived for various sorghum cultivars. Regression equations from pooled data were used to obtain estimates of equilibrium density and maximum reproductive rate, and these estimates were used to construct models expressing nematode Pf across a range of initial densities. Many of these models were robust, encompassing a range of sites, season, crop cultivars, and planting dates. Quadratic models derived from pooled field data provided an alternative method for expressing Pf as a function of Pi.     

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.     

New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina

We report an additional case of long-term persistence of Paranosema locustae in grasshoppers of Argentina. The pathogen was introduced from North America on rangeland at Loncopué, Neuquén province. Microsporidia were not detected in pre-introduction samples whereas infected grasshoppers were found 11 years after introduction. Affected grasshoppers were the melanoplines Dichroplus elongatus, Dichroplus maculipennis, and Scotussa lemniscata, some of them with high spore loads. The case highlights the ability of P. locustae to recycle in local grasshopper communities by parasitizing susceptible species other than the natural hosts.     

Comparative studies on the activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in the leaves of triticale and wheat infected with Stagonospora nodorum

The activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase, constitutive and induced by Stagonospora nodorum were examined in the 10 – 14 day old seedlings of three triticale and two wheat cultivars under controlled environmental conditions and in flag leaves of two triticale cultivars in the field. Two S. nodorum isolates of different virulence were used. Both the constitutive and induced activities in triticale and wheat depended on genotype and in triticale the effect of growth conditions was also evidenced. The constitutive activities of chitinase, β-1,3-glucanase and peroxidase were several fold lower in flag triticale leaves in plants from the field than in the seedlings, growing under controlled conditions, but induction in the infected flag leaves was significantly more pronounced. In triticale genotypic differences in the response to infection were revealed only upon inoculation by S. nodorum isolate of higher virulence. The enzymatic activities increased several fold during successive days after the infection except for phenylalanine ammonia lyase. Induction of this enzyme was only transient and the activity decreased 48 or 96 h after infection when the activities of other enzymes were rising. In flag leaves in the field this activity was differentiated only after infection with more a virulent strain. A tendency appeared in triticale seedlings for association of the resistance to the pathogen with lower enzymatic constitutive activities. This relationship became more evident in triticale infected by S. nodorum and may imply that although the investigated enzymes are certainly involved in general, non-specific defense mechanism, they do not decide on the resistance to pathogen at least in the early stages of infection and cooperate with other factors in the complex pathogen-plant interaction. One can also assume that the enzymatic activities are associated with severity of infection rather than resistance to pathogen.     

Transmission of Vairimorpha invictae (Microsporidia: Burenellidae) infections between red imported fire ant (Hymenoptera: Formicidae) colonies

Red imported fire ant, Solenopsis invicta, colonies were successfully infected with the microsporidium Vairimorpha invictae by introducing live larvae, pupae, or dead adults from V. invictae-infected field colonies collected in Argentina. Introductions with 4th instar larvae or non-melanized pupae obtained from infected field colonies, resulted in infection of 40% of the inoculated colonies. Introductions of 4th instars or melanized pupae produced from colonies that were initially infected in the laboratory, resulted in infections of 83% of the colonies, thus perpetuating the infection in other colonies. Infection was detected in 2 of 6 colonies after introducing adult worker caste ants that had died with V. invictae. The average number of adults and the volume of immature ants per colony were significantly lower in the infected than in the control colonies. Infected colonies had 86% fewer adults per colony and 82% less immature ants than the controls. A portion of the 16S rRNA gene of the V. invictae identified from these studies was amplified, cloned, and sequenced; the 1251 nucleotide amplicon was 100% identical to the 16S rRNA gene sequence recorded previously in the GenBank database, thus verifying the species as V. invictae. This is the first report of the artificial transmission of this pathogen to uninfected ant colonies, and demonstration of its ability to hinder growth in individual colonies.     

Pathogenicity of the pinewood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings

Pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings was tested with inoculation experiments under nursery conditions. Water suspensions of nematodes (mixed stages cultured on Botrytis cinerea or dispersal fourth-stage juveniles (DJ4) extracted from the adult Japanese pine sawyer, Monochamus alternatus) were injected into the stems of 2- and 3-year-old Japanese larch and Japanese black pine, Pinus thunbergii, seedlings growing in a nursery. In another treatment, Japanese pine sawyer adults holding DJ4 were released under a net that covered the upper half of the seedlings. Regardless of nematode inoculation method, Japanese larch seedlings were as susceptible as Japanese black pine seedlings to B. xylophilus under nursery conditions. The rate of disease development was similar on larch and pine seedlings. Nematode population densities were lower in the stems of dead larch seedlings than in the stems of dead pine seedlings. Histopathological observations revealed that the distribution of nematodes in the stems of dead larch seedlings was mostly limited to the cortex, phloem and cambial zone. Traumatic resin canal formation was one of the most characteristic symptoms in larch seedlings which was dissimilar to that in pine seedlings.     

Post-Infection Development and Histopathology of Meloidogyne incognita in Resistant Cotton

The numbers of Meloidogyne incognita larvae which migrated from cotton roots declined over a 16-day period, but the difference in numbers migrating from resistant and susceptible cultivars was not significant. Larvae penetrated susceptible roots, matured, and reproduced within 14 days following inoculation, whereas nematode development in the resistant roots was greatly retarded. Three types of histological responses were observed in infected, resistant roots, and these correlated with the degree of nematode development. Some galls were examined which contained only fragments of nematodes; others contained no detectable traces of developing larvae. Formation of druses in galls, but not in healthy tissue, was noted in both cultivars 20 days after inoculation. Massive invasion of roots resulted in deep longitudinal fissures of root cortex.     

Attenuation of fungal infection in thermoregulating Locusta migratoria is accompanied by changes in hemolymph proteins

Hemolymph proteins in the locust, Locusta migratoria migratorioides infected with the fungus Metarhizium anisopliae var acridum were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under conditions that allowed locusts to thermoregulate, 2 proteins, ITB1 (ca. 18kDa) and ITB2 (ca. 13kDa) were induced 48h post inoculation. In contrast, under non-thermoregulating conditions, only 1 band, INTB1 (ca. 18kDa) was induced with similar molecular mass to ITB1. ITB1 and ITB2 were N-terminally sequenced but showed little homology to known proteins. The induction of hemolymphal proteins in infected, thermoregulating locusts and implication in insect immune defence are discussed.