Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Comparison of AC electronic monitoring and field data for estimating tolerance to Empoasca kraemeri (Homoptera: Cicadellidae) in common bean genotypes

Two methods for estimating the tolerance of common bean genotypes to Empoasca kraemeri Ross & Moore were compared, using a yield trial carried out at Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, versus stylet penetration tactics measured by AC electronic feeding monitors. A stylet penetration index was devised based on principal component scores of three penetration tactics identified (pulsing laceration, cell rupturing, and lancing sap ingestion), combined with knowledge of the hopperburn symptoms caused by each tactic. Tolerant genotypes, as classified by the CIAT yield index, showed significantly more unprotected yield and lower hopperburn scores than the susceptible control. They also induced performance of less pulsing laceration (the tactic considered most damaging to the plant), and more of the other two, mitigating tactics, especially cell rupturing. When index values were calculated for each genotype, stylet penetration index values matched those of the yield index for three out of five genotypes: two EMP-coded tolerant lines ('EMP 385' and 'EMP 392') and the susceptible control 'BAT 41'. Thus, for these three genotypes, all subsequent hoppereburn symptoms are predictable by the type of feeding behavior performed on them. 'Porrillo Sintético' and 'EMP 84', considered borderline genotypes by the yield index, were overestimated and underestimated respectively, by the stylet penetration index. We postulate that, for these two genotypes, plant physiological responses to feeding (either compensatory or heightened sensitivity, respectively) synergize with type of feeding performed to generate the overall hopperburn condition. This multivariate analysis of electronic monitoring data was successfully used to devise an index of resistance. The implications of using the stylet penetration index and the advantages of using electronic monitoring in a bean-breeding program are discussed.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.     

Laboratory-and Field-Phenotyping for Drought Stress Tolerance and Diversity Study in Lentil (<i>Lens culinaris</i> Medik.)

Drought susceptibility and low genetic variability are the major constraints of lentil (Lens culinaris Medik.) production worldwide. Development of an efficient pre-field drought phenotyping technique and identification of diversified drought tolerant lentil genotype(s) are therefore vital and necessary. Two separate experiments were conducted using thirty diverse lentil genotypes to isolate drought tolerant genotype(s) as well as to assess their diversity. In both of the experiments, significant (p ≤ 0.01) variation in genotype (G), treatment (T) and G X T was observed for most of the studied traits. In experiment I, genotypes were examined for drought tolerance at the seedlings stage under hydroponic conditions by assessing root and shoot traits. Among the 30 genotypes studied, BM-1247, BM-1227 and BM-502 were selected as highly tolerant to drought stress as they showed maximum seedling survivability and minimum reduction in growth parameters under drought stress. In experiment II, the genotypes were assayed for diversity and drought stress tolerance based on morphological traits grown under field condition. Drought stress caused a substantial reduction in yield attributing traits, however, the genotypes BM-1247, BM-981, BM-1227 and BM-502 were categorized as drought tolerant genotypes with less than 20% yield reduction. The field screening result of drought stress tolerance was coincided well with the results of laboratory screening. Genetic divergence study reflected the presence of considerable diversity among the genotypes. Considering laboratory and field screening results, the genotypes, BM-1247, BM-1227, BM-981 and BM- 502 were selected as the best drought tolerant genotypes. This information can be exploited for further breeding in developing drought tolerance in lentil.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Effect of temperature and sources of carbon and nitrogen on the conidial germination and appresoria formation in Colletotrichum capsici

The effect of temperatures and exogenous supply of different carbon and nitrogen sources on the conidial germination and appresoria formation inC. capsici has been studied. 25 °C was observed to be the best temperature for conidial germination. At 18 °C, conidia germinated only in hanging drops and did not germinate on 2 % agar. Amongst carbon sources, 1-rhamnose supported maximum conidial germination and appresoria formation. Potassium nitrate supported very good conidial germination and appresoria formation. Ammonium nitrate, ammonium sulphate and ammonium chloride were found to be inhibitory. A few aminoacids stimulated conidial germination but dl-alanine, dl-methionine, tyrosine and l-glutamine were found to be inhibitory.     

Time of sowing and temperature effects on the flowering of Stylosanthes guianensis

Seedlings of Stylosanthes guianensis var. guianensis cv. Cook and of two selections of S. guianensis var. pauciflora (CIAT 1280 and CIAT 1062) were grown at day/night temperatures of 20°/25°, 30°/25°, and 35°/30°C in a naturally-lit glasshouse at latitude 27°30'S. Sowings were made in decreasing daylength at 30-day intervals from 22 January to 21 May. Cv. Cook and the CIAT 1280 selection did not flower fully if sown after 22 January and the CIAT 1062 selection did not flower if sown after 22 March. This is interpreted as a long-short day flowering response. Usually, flowers were initiated earlier and at a lower node at 25?/20°C than at the warmer temperatures. At 25?/20°C the first flowers to appear were produced predominantly at the terminal apex of the main stem in cv. Cook and the CIAT 1062 selection, but not in the CIAT 1280 selection. At the two warmer temperatures first flowers were more commonly produced at the terminal apex of primary, secondary and tertiary branches. There were more inflorescences per plant on earlier than later sown plants when measured 21 days from appearance of the first flower and the most inflorescences were produced by cv. Cook at 25?/20°C, by selection CIAT 1062 at 30°/25°C, and by CIAT 1280 at 35°/30°C.     

Canola (Brassica napus) germplasm shows variable allelopathic effects against annual ryegrass (Lolium rigidum)

Aims

The allelopathic activity of canola (Brassica napus) germplasm was investigated using the important Australian weed, annual ryegrass (Lolium rigidum) as the target species.

Methods

Three different canola plant densities (10, 20, and 30 seedlings/beaker) of each of 70 world-wide genotypes were tested in vitro in close proximity to annual ryegrass seedlings.

Results

The allelopathic activity of canola, as measured by reduction in annual ryegrass root and shoot growth, increased with canola crop seedling densities. Density did not consistently influence shoot length of annual ryegrass. Greater shoot length suppression was observed in genotype cv. Rivette and BLN3343CO0402. The Australian genotype cv. Av-opal and the breeding line Pak85388-502 suppressed root length of ryegrass more than other genotypes, even at low densities. At the lowest density, the least allelopathic genotypes were cv. Barossa and cv. Cescaljarni-repka, although they became more allelopathic at higher density. An overall inhibition index was calculated to rank each of the canola genotypes. There were significant differences between canola genotypes in their ability to inhibit root and shoot growth in ryegrass.

Conclusion

Considerable genetic variation exists among canola genotypes for their allelopathic effects on annual ryegrass. Further investigation is required to determine the allelopathic mechanisms, particularly to identify the responsible allelochemical(s) and the gene(s) controlling the trait. This research suggests that highly allelopathic canola genotypes can be potential for controlling weeds such as annual ryegrass in integrated weed management programs.     

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Summary Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.     

Response of resistant and susceptible Brachiaria spp. genotypes to simultaneous infestation with multiple species of spittlebugs (Hemiptera: Cercopidae)

The response of one susceptible and three resistant Brachiaria spp. (Hemiptera: Cercopidae) genotypes to individual or combined attacks by nymphs of Aeneolamia varia (F.), Aeneolamia reducta (Lallemand), Zulia carbonaria (Lallemand), and Zulia pubescens (F.) was studied. We assessed the effect of infesting plants of the susceptible check BRX 44-02 and of the A. varia-resistant genotypes CIAT 6294 and CIAT 36062 with A. varia, Z. carbonaria, or Z. pubescens either alone or in two-species combinations. In a second trial, we studied the performance of BRX 44-02, CIAT 6294, and the multiple resistant clone SX01NO/0102 exposed to individual or combined attack by A. reducta and Z. carbonaria. In a third trial, we compared the response of BRX 44-02, CIAT 6294, and CIAT 36062 to individual A. varia, Z. carbonaria, or Z. pubescens attack as opposed to a combined three-species attack. Plant damage scores and percentage of nymphal survival were recorded in all three trials. Data on percentage of survival indicated that competition between and among spittlebug species occurs. However, we found no evidence of interaction between species competition and different levels of resistance to spittlebug. Rather, host genotype reactions conformed to previously known categories of resistance regardless of the presence of more than one spittlebug species. Resistance rather than competition seems to have been the overriding factor determining nymph survival and resistance expression (damage scores) in these experiments. Our results corroborate the need to develop brachiariagrass genotypes with multiple resistance to spittlebugs.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Infection biology and defence responses in sorghum against Colletotrichum sublineolum

Aims:  To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes.
Methods and Results:  Infection biology and defence responses were studied quantitatively by light microscopy, H₂O₂ accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H₂O₂, HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H₂O₂ accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration.
Conclusions:  The early accumulation of H₂O₂ and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis.
Significance and Impact of the Study:  The fact that there is a significant correlation between induced accumulation of H₂O₂, papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.     

Prepenetration and Penetration of Colletotrichum gloeosporioides into Guava Fruit (Psidium guajava L.): Effects of Temperature,Wetness Period and Fruit Age

In this study, we determined the influences of temperature, wetness period and guava fruit age on infection caused by Colletotrichum gloeosporioides. Optimal temperatures in vitro for germination, appressoria formation and melanization were 22.7, 20.6 and 23°C, respectively. In vivo, the optimal temperatures for germination and appressoria formation were 22.5 and 23°C, respectively. Values for germination, appressoria formation and melanization were higher as the wetness period increased. There was no difference in conidial germination and appressorial formation on fruit of different ages. On the surface of 10‐, 35‐ and 60‐day‐old fruit, despite the high percentage of appressorial formation, there was no development of the penetration peg. Penetration pegs were only observed on the 85‐ and 110‐day‐old fruit. Thickness of the cuticle, size and architecture of epidermal and parenchymal cells, as well as the content of phenolic compounds changed as the fruit ripened.     

Epicatechin and catechin may prevent coffee berry disease by inhibition of appressorial melanization of Colletotrichum kahawae

Colletotrichum kahawae is the causal agent of coffee berry disease. Appressorial melanization is essential for the fungal penetration of plant cuticle. Epicatechin is abundant in green coffee berry pericarp. Inoculation of C. kahawae conidial suspension containing 1.2 mg epicatechin or catechin/ml did not affect conidial germination or appressorial formation but appressorial melanization was completely inhibited and infection by the treated conidia was less than 30% of the untreated control. Epicatechin and catechin may, therefore, prevent coffee berry disease by inhibition of the appressorial melanization of C. kahawae.     

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens

 The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed. Received: 5 April 1999 / Revision received: 13 September 1999 / Accepted: 6 October 1999     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Nitrogen nutrition influences some biochemical responses to iron deficiency in tolerant and sensitive genotypes of Vitis

The effects of nitrogen source on iron deficiency responses were investigated in two Vitis genotypes, one tolerant to limestone chlorosis Cabernet Sauvignon (Vitis vinifera cv.) and the other susceptible Gloire de Montpellier (Vitis riparia cv.). Plants were grown with or without Fe(III)-EDTA, and with NO₃⁻ alone or a mixture of NO₃⁻ and NH₄⁺. Changes in pH of the nutrient solution and root ferric chelate reductase (FC-R) activity were monitored over one week. We carried out quantitative metabolic profiling (¹H-NMR) and determined the activity of enzymes involved in organic acid metabolism in root tips. In iron free-solutions, with NO₃⁻ as the sole nitrogen source, the typical Fe-deficiency response reactions as acidification of the growth medium and enhanced FC-R activity in the roots were observed only in the tolerant genotype. Under the same nutritional conditions, organic acid accumulation (mainly citrate and malate) was found for both genotypes. In the presence of NH4⁺, the sensitive genotype displayed some decrease in pH of the growth medium and an increase in FC-R activity. For both genotypes, the presence of NH₄⁺ ions decreased significantly the organic acid content of roots. Both Vitis genotypes were able to take up NH₄⁺ from the nutrient solution, regardless of their sensitivity to iron deficiency. The presence of N-NH₄⁺ modified typical Fe stress responses in tolerant and sensitive Vitis genotypes.     

Parental effects in Lychnis flos-cuculi. I: Seed size,germination and seedling performance in a controlled environment

Selection responses in natural plant populations depend on how the phenotypic variation of traits is composed. The contributions of nuclear genetic, maternal, paternal, environmental and inbreeding effects to variation in time to germination, germination percentage, and seed- and seedling size were studied in a population of Lychnis flos-cuculi. It was found that: (1) Maternal effects predominated in the determination of progeny seed size and germination characteristics; (2) Maternal environment during seed development was less important than maternal genotype; (3) Small but significant variation within maternal families could be observed among individuals sired by different fathers; (4) Additive genetic variance was significant for seedling size 4 weeks after germination. In conclusion, selection shortly after emergence will mainly favour particular maternal genotypes, while selection later in the life cycle may act upon zygotic genotypes. Inbreeding depression was significant, especially for vegetative growth. Consistent differences were found among maternal genotypes in the degree of variation in the time to germination, suggesting that selection could operate to favour polymorphic or uniform germination behaviour.