Effect of Didymella applanata (Niessl/Sacc.) on proline and hydroxyproline containing cell wall proteins of red raspberry

Structural cell wall proteins and their immobilisation through formation of covalent cross-links belong to important defense response mechanisms. In this work, the D. applanata — induced increase of wall-bound proline and hydroxyproline contents were associated with red raspberry resistance. The effect did not depend on the developmental stage and lignification degree of infected primocanes. Pathogen-induced accumulation of hydroxyproline noted in susceptible plants suggested the post-translational hydroxylation of proline-rich proteins and/or may be of other wall proteins. Moreover, the developmentally related increase of hydroxyproline content was associated with the resistance acquirement noted at the end of the summer period. The increase of cell wall structural protein levels was accompanied by free proline accumulation in the cytosol fraction.     

The Effect of Chitinase on Didymella applanata, the Causal Agent of Raspberry Cane Spur light

In vitro and in vivo studies were conducted to assess the efficacy of the two microbial chitinases Chi I (from Streptomyces sp.) and Chi II (from Serratia marcescens) on Didymella applanata (Niessl.) Sacc., the fungus which causes spur blight of raspberry (Rubus idaeus L.). D. applanata was isolated from canes of diseased raspberries in a plantation in Novosibirsk, Russia. In vitro, the effective concentration of Chi I that reduced the growth of D. applanata was 0.4 U/ml (p = 0.05), but Chi II had no influence on the growth of the fungus in medium. In inoculation experiments on raspberry canes, both chitinases at the rate 0.5 U/ml reduced fungal development. In plantation where canes were inoculated after spraying with chitinase, fruiting bodies of fungus failed to form in all enzyme treatments, whereas a significant number of these fungal fruiting bodies (12.8 per cm²) developed in control treatments lacking chitinases spraying. The chitinases reduced the size of lesions and limited the infection of internal tissues of canes. Field testing of Chi I under natural conditions showed a significant suppression of the independent spur blight. These studies form the basis for further evaluation of ecologically benign control measures for raspberry spur blight.     

Phenolic Compounds and the Polyphenoloxidase and Peroxidase Activity in Callus Tissue Culture-Pathogen Combination of Red Raspberry and Didymella applanata (Niessl.) Sacc.

The content of phenolic compounds as well as the activity of polyphenoloxidase and peroxidase in the red raspberry callus tissue after D. applanata infection were investigated. Two red raspberry cultivars: Latham — relatively resistant and M. Promise — susceptible were used. In the M. Promise cultivar the amount of phenols, especially o-diphenols, decreased after infection and stayed more or less on the same level in Latham cultivar. The activity of polyphenoloxidase increased in both of the investigated cultivars, however in the tissue of Latham cultivar this rise was more pronounced than in the M. Promise one. The changes in peroxidase activity after infection were inconsiderable. The relation between the changes observed after infection in the phenolic metabolism and the differentiated susceptibility of callus tissue to D. applanata of the investigated cultivars was discussed. The obtained results explain partially the browning of infected callus tissue of one of the investigated cultivar i.e. M. Promise which had already been observed in the earlier investigations.     

Field testing of BACTICIDE®, PHYTOVERM® and CHITINASE for control of the raspberry midge blight in Siberia

Three products based on compounds of biological origin were tested for their ability tocontrol the raspberry midge blight in theSiberian region of Russia. Bacillusthuringiensis subsp. israelensis(BACTICIDE^®) and Streptomycesavermitilis metabolites (PHYTOVERM^®) were used against Thomasiniana theobaldi (ageneral member of the midge blight) andCHITINASE was used against fungi (mainly Didymella applanata) associated with T. theobaldi. The BACTICIDE^®(0.2%) and PHYTOVERM^® (0.2%) sprays caused a 2-fold decrease in midge blight severity and the sameeffect was obtained with chemical insecticides.The CHITINASE (1%) spray caused a 4-folddecrease in the severity of midge blight. Inaddition, CHITINASE and PHYTOVERM^®caused a significant suppression of theindependent spur blight. These studies form thebasis for further evaluation of ecologicallysafe control of the raspberry midge blight.     

Insect feeding on spores of a bracket fungus, Elfvingia applanata (Pers.) Karst. (Ganodermataceae, Aphyllophorales)

Insects visiting sporocarps of Elfvingia applanata, a wood-rotting bracket fungus, were examined in Kyoto, central Japan. Mycodrosophila flies (Drosophilidae: Diptera) were predominant and visited the spore-producing sporocarps exclusively. They were observed feeding on the spores, and a number of spores seemed to be alive even after having passed through insects digestive tracts. In addition, the insects attached a number of spores on their body surfaces. In a rearing experiment with insects caught from E. applanata sporocarps, Mycodrosophila flies excreted 7700–469 000 and dropped 10–000–329 000 of viable spores during 48 h after collection. They were supposed to migrate among the sporocarps of other bracket fungi growing on different logs or stumps, suggesting that Mycodrosophila flies may act as spore-dispersal agents for E. applanata.     

Effectiveness of resistance genes to the large raspberry aphid, Amphorophora idaei Börner, in different raspberry (Rubus idaeus L.) genotypes and under different environmental conditions

The introduction into commerce of raspberry cultivars with major gene resistance to the large raspberry aphid, Amphorophora idaei, an important pest and virus vector on red raspberry in Europe, has been very effective both in decreasing pest numbers and greatly restricting infection with the viruses it transmits. However, biotypes of the aphid able to overcome these genes have developed in the field in recent years. Additionally, in field and laboratory tests, the response to aphid biotypes and recognised aphid strains of certain raspberry cultivars, such as Glen Prosen and Delight, differ markedly despite the fact that they are reputed to contain the same A. idaei‐resistance gene, A₁. In attempts to understand the reasons for this difference in response, analysis was made of the segregation of progeny seedlings from crosses between A. idaei‐resistant and ‐susceptible cultivars to two recognised strains of the aphid. These studies showed that, as expected, cv. Autumn Bliss contained the A. idaei‐resistance gene, A₁₀, and cvs Delight and Glen Prosen each contained the A. idaei‐resistance gene, A₁. When progeny seedlings were assayed in a heated glasshouse as young plants and in an unheated Tygan house as 1 m tall plants, the segregation ratios for resistance and susceptibility to A. idaei were largely unchanged. However, when the resistance of individual progeny plants was assessed, c. 37% of the putative gene A₁‐containing progeny and 9–23% of the putative gene A₁₀‐containing progeny, behaved differently in these two environments. Experiments involving an A. idaei‐resistant and ‐susceptible parent cultivar showed that shading plants increased their susceptibility to A. idaei colonisation. Whilst this shading effect has implications for experimentally detecting A. idaei‐resistant progeny in segregating raspberry seedlings, it does not explain the difference in field resistance to A. idaei of cvs Delight and Glen Prosen. Such differences in the field seem best explained by the presence in these cultivars of ‘minor’ genes for A. idaei resistance and/or susceptibility that influences the effectiveness of gene A₁.     

Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.     

Biology of the European large raspberry aphid (Amphorophora idaei): its role in virus transmission and resistance breakdown in red raspberry

1 The European large raspberry aphid Amphorophora idaei Börner is the most important vector of viral diseases afflicting commercially grown red raspberry ( Rubus idaeus L.) in Northern Europe, with European raspberry production amounting to 416 000 tonnes per annum. This review synthesizes existing knowledge on its biology and interactions with other organisms, including its host plant and the viral pathogens it vectors.
2 Information about trophic interactions with other insect herbivores and natural enemies is reviewed. Vine weevils Otiorhynchus sulcatus compromise aphid resistance in some raspberry cultivars, increasing A.   idaei abundance by 80%. Parasitoids show mixed success in parasitizing A.   idaei , although Aphidius ervi attack rates more than doubled when A.   idaei fed on a partially susceptible raspberry cultivar, compared with a resistant variety. These findings are discussed in the context of potential biological control as part of an integrated pest and disease management framework.
3  Amphorophora idaei transmits four known viruses: Black raspberry necrosis virus, Raspberry leaf mottle virus, Raspberry leaf spot virus and Rubus yellow net virus , with A.   idaei taking as little as 2 min to transmit some viruses.
4 Existing control strategies, including resistant cultivars, insecticides and eradication of disease from parent plants, are described. In particular, strong selection pressures have resulted in A .  idaei overcoming genetic resistance in many raspberry cultivars and most insecticides are now ineffective.
5 Future directions for the sustained control of A.   idaei are suggested, taking into consideration the possible effects of climate change and also changes in agronomic practices in U.K. agriculture.     

Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.)

Two dominant genes conferring complete resistance to specific isolates of the rice blast fungus, Pyricularia grisea Sacc., were located on the molecular map of rice in this study. Pi-l(t) is a blast resistance gene derived from the cultivar LAC23. Its map location was determined using a pair of nearly isogenic lines (NILs) and a B₆F₃ segregating population from which the isoline was derived. RFLP analysis showed that Pi-l(t) is located near the end of chromosome 11, linked to RZ536 at a distance of 14.0±4.5 centiMorgans (cM). A second gene, derived from the cultivar Apura, was mapped using a rice doubled-haploid (DH) population. This gene was located on chromosome 12, flanked by RG457 and RG869, at a distance of 13.5+-4.3 cM and 17.7+-4.5 cM, respectively. The newly mapped gene on chromosome 12 may be allelic or closely linked toPi-ta. (=Pi-4(t)), a gene derived from Tetep that was previously reported to be linked to RG869 at a distance of 15.4±4.7 cM. The usefulness of markers linked to blast resistance genes will be discussed in the context of breeding for durable blast resistance.     

小立碗藓GPX家族的功能分化与催化特性研究

谷胱甘肽过氧化物酶(GPX)在植物抵抗氧化胁迫中发挥重要作用。该研究从小立碗藓(Physcomitrella patens)基因组中挖掘到3个GPX基因,分别命名为PpGPX1、PpGPX2和PpGPX3。其中PpGPX1和PpGPX3只含有1个外显子,而PpGPX2含有6个外显子。表达模式分析发现PpGPX1和PpGPX2在检测的所有条件下均表达,而PpGPX3在检测的所有条件下均不表达。蛋白亚细胞定位分析发现,PpGPX1蛋白定位在细胞质,而PpGPX2蛋白定位在叶绿体。在大肠杆菌中表达并纯化了PpGPX1和PpGPX2蛋白,酶学性质分析发现,PpGPX1和PpGPX2蛋白均只能利用Trx电子供体系统,而不能利用GSH电子供体系统;PpGPX2蛋白对过氧化物底物的催化活性和催化效率均高于PpGPX1。基因结构、表达模式、亚细胞定位和蛋白酶学性质的差异预示小立碗藓GPX基因家族成员发生了功能分化,将PpGPX2蛋白的Pro158、Phe167和Phe172氨基酸残基均突变为Ala,发现突变体蛋白对底物催化活性降低,说明这3个氨基酸位点对PpGPX2蛋白具有重要催化活性。     

Use of Brassica callus culture to induce sporulation in Alternaria brassicae (Berk.) Sacc.

A non-sporulating isolate of Alternaria brassicae, inoculated on callus culture of Brassica juncea cv. Kranti, colonized the callus and produced spores. When captafol, a fungicide, was added (100 mg/l) to the callus culture medium, if effectively checked fungal contamination and saprophytic growth of A. brassicae on culture medium, without adversely affecting callus growth or establishment of dual culture.     

Comparison of rice lines derived through anther culture and the pedigree method in relation to blast (Pyricularia grisea Sacc.) resistance

Crosses were made between Fanny (highly susceptible to blast) and 11 cultivars differing in blast resistance. Using the pedigree method (PM) segregating generations were evaluated and selected for blast resistance. Via anther culture (AC), doubled-haploids were obtained from F₁ plants and from F₂ blast-susceptible plants. Pedigree and anther culture-derived lines were planted together and evaluated for blast resistance under rainfed conditions at the Santa Rosa Experiment Station, Villavicencio, Colombia. The principal objective was to compare PM and AC in terms of their efficiency in producing rice lines resistant to blast. Results of a stratified analysis showed an association between method and blast resistance. Results of the logit-model analysis showed that AC produced a significantly (P=0.0001) higher proportion of lines with initial blast resistance (leaf- and neck-blast reaction 4) than did PM across all cross types. Stable blast resistance was assessed based on field performance over 3 years. AC was superior to PM in generating stable resistance for only some cross types. Consequently, with a few exceptions, AC can be used as effectively as PM to develop rice cultivars resistant to blast, with savings in time and labor. Additionally, blast-resistant lines were obtained either by the pedigree method or by anther culture from crosses between blast-susceptible cultivars (Fanny/CICA4 and Fanny/Colombial). This excludes somaclonal variation as a possible mechanism responsible for this resistance and suggests that a recombination of minor genes could have occurred and was fixed through either method. However, the stability of the resistance was greater in pedigree-derived lines. The implications of these findings for rice blast-resistance breeding are discussed.     

Growth pattern and cellular nitrogen and phosphorus contents of the dinoflagellate Peridinium penardii (Lemm.) Lemm. causing a freshwater red tide in a reservoir

The population growth pattern and related changes in both the nitrogen and phosphorus contents in the cell of the dinoflagellate Peridinium penardii (Lemm.) Lemm., which formed a freshwater red tide in a reservoir, were studied in situ. An exponential increase with time in population density was found. A specific growth rate of 0.25 d^–1 was observed. The cellular content of phosphorus per cell decreased from 6.0 × 10^–5 µg to 9.2 × 10^–6 µg along with an increase in population density from 8.0 × 10² cells ml^–1 to 2.5 × 10⁴ cells ml^–1. A prominent change in the cellular nitrogen did not occur. Decreasing cell content and continuous uptake of phosphorus were advantageous for P. penardii to form a freshwater red tide under P-limited conditions.     

Hemocytic defense response to the entomopathogenic fungus Beauveria bassiana in the migratory grasshopper Melanoplus sanguinipes

Hemocytic defense response of the migratory grasshopper, Melanoplus sanguinipes (Fab.), to conidia of the white muscardine fungus, Beauveria bassiana (Bals.) Vuill., was studied using phase-contrast photomicroscopy, hemocyte counting and hemocyte aggregation or nodule formation. Grasshoppers were injected with an aqueous suspension of conidia. Adherence of B. bassiana conidia to granulocytes occurred within 10 min and the conidia were encapsulated by these hemocytes 6 h postinjection. Hemocytopenia was accompanied by nodule formation after injection of B. bassiana conidia into grasshoppers. The conidia germinated within the nodules and grew into hyphal forms within the hemolymph 12 to 24 h postinjection. We conclude that B. bassiana competes well with nodule formation by hemocytes of M. sanguinipes and often escapes complete encapsulation.

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Résumé Le mécanisme de défense du hémocyte de la sauterelle migratrice Melanoplus sanguinipes envers le champignon pathogène Beauveria bassiana a été etudié a l'aide du photomicroscope a contrase de phase, par dénombrement des hemocytes, ainsi que des nodules formées par l'agrégation des hémocytes. L'adhérence des conidies de B. bassiana aux hemocytes a été observée 10 min après l'injection et leur encapsulement après 1 h. Une baisse des taux d'hémocytes a fait suite a la formation de nodules apres l'injection de conidies dans les sauterelles. Après le déclin initial du taux des hémocytes, une hausse s'est produite dans les sauterelles auquelles on a injecté 10⁶ conidies. Les conidies ont germé dans les nodules et la croissance du mycélium s'est produite dans l'hémolymphe 24 h après avoir injecté. Cette étude a revelé que M. sanguinipes peut exercer temporairement un mécanisme de défense cellulaire contre des conidies fongiques a une concentration de 10⁶ conidies.

     

Flowering and gibberellins in a mutant red clover (Trifolium pratense L.)

Relationships between gibberellins and floral initiation were investigated in a conditional non-flowering mutant of red clover, Trifolium pratense. Untreated mutant plants will not flower under long-days, but will do so when certain GAs are applied. Gibberellins, A₃, A₁, A₇, and A₅ all resulted in both stem elongation and flowering whilst GA₄ produced the elongation only. Applications of GA₂₀, GA₈ and GA₁₃ under long-days had no detectable effect. Thus, by combining the use of the mutant with the application of different GAs, the correlation between the processes of stem elongation and floral initiation, which is normally strongly expressed in this species, was broken. Endogenous gibberellins shown to be present in normal plants were also found in the mutant genotype. Gibberellins alone were not sufficient to initiate floral development in the mutant, there being an essential element of interaction with long-days. These results are discussed in relation to the nature of the lesion in the mutant and the signal provided by the applied gibberellin.     

Intra- and interspecific inheritance of some components of the resistance to leaf rust (Melampsora larici-populina Kleb.) in poplars

Clonal variation for rust resistance was studied in a factorial mating design of Populus trichocarpa and P. deltoides with 17 intraspecific and 15 interspecific progenies. Susceptibility to Melampsora larici-populina was assessed both under natural conditions in the nursery and after leaf-disk inoculation with two pathogen races in a controlled environment. Different components of the resistance were taken into account: immunity in the field or in the laboratory, latent period, number and size of uredia in the laboratory, and field resistance at the end of the growing season. Genes controlling immunity were contributed only by the P. deltoides parents. The distributions of clonal means within each family suggested a polygenic inheritance of field resistance in the P. trichocarpa x P. trichocarpa crosses, but major gene effects were suspected in the interspecific progenies. Inefficiency of the quantitative mechanisms of resistance in the interspecific F₁ hybrids might have important implications for future breeding strategies. Field and laboratory trials complemented each other well, and a combined selection approach is proposed.     

Relationship between soluble carbohydrate level and tolerance of meadow fescue callus to Bipolaris sorokiniana (Sacc.) Shoem. and Drechslera dictyoides (Drechsl.) Shoem. metabolites

The aim of the presented work was the search for the relationship between the level of soluble carbohydrates in callus tissues of eight meadow fescue (Festuca pratensis Huds.) cultivars and their growth ability on media containing Bipolaris sorokiniana and Drechslera dictyoides metabolites. Calli were induced from mature grains using the method previously described (Płażek 1994). Callus obtained from single caryopsis was cut into three pieces which were weighted and put on the media with or without pathogen metabolites. Tissue selection was performed by means of “double-layer culture” technique (Lepoivre et al. 1986). After two-week culture in the darkness at temp. of 25°C the calli were weighted again. The sugar level in tissue was measured by means of colorimetric method of Klein & Weissman according to Snell (1961). Fresh mass decrease of calli developing on the media with fungus metabolites was observed by all studied object. The tolerance of calli of the tested cultivars to metabolites of both pathogens was significantly different. However, significant similarity between the tolerance of calli of particular varietes to both fungi was noted. The soluble carbohydrate contents in control tissue of all studied cultivars were similar and their values ranged between 2.4 and 3 % of fresh mass. B. sorokiniana metabolites caused a significant decrease of the sugar content in calli, while D. dictyoides metabolites did not decrease the sugar level.     

Fixation of 14CO2 in tissue cultured red raspberry prior to and after transfer to soil

The ¹⁴CO₂ uptake of an aseptically cultured red raspberry clone (Rubus ideaus L.) was examined prior to and after transfer to soil. Individual leaves of transplants, both persistent from culture and new ones, were tested 5 weeks after transplant for ¹⁴CO₂ uptake capability. Transplant leaves of successive weekly age classes took up ¹⁴CO₂ at increasing rates per unit area, displaying a spectrum of photosynthetic competence from low levels close to that of leaves from culture, to that of control plants. This is illustrative of acclimatization to the soil environment and was related to transplant light intensity.     

Apple maggot fly response to red sphere traps in relation to fly age and experience

Responses of the apple maggot fly,Rhagoletis pomonella (Walsh), to 8 cm red sticky sphere traps, baited with the synthetic fruit odor butyl hexanoate, were investigated in field-caged apple trees containing green or red Gravenstein apples. Trap capture rate and the probability of oviposition in apples before capture generally increased with female age and number of mature eggs in ovaries. Two days of pre-test exposure of mature females to red hawthorns, green Red Delicious apples or green Gravenstein apples had no significant effect on the likelihood of a fly finding a red sphere. However, before capture on a sphere or departing a tree, hawthorn-exposed females found significantly fewer apples and laid significantly fewer eggs than females exposed to Gravenstein apples. Variation in duration of pre-test exposure (1–4 days) of flies to Gravenstein apples had no detectable influence on female response to apples or to a red sphere in a test tree. The relevance of these findings to effectiveness of sphere traps, forR. pomonella control in commercial orchards is discussed.