Common Leafspot of Alfalfa: Ascospore Germination and Disease Development in Relation to Moisture and Temperature

In a moist chamber Pseudopeziza medicaginis ascospores infected alfalfa (Medi sativa L.) moderately to abundantly within 6–10 h at 10–20 °C and within a longer time-span outside this temperature range. Approximate limits of the range were 2.5 and 28 °C; no infection took place at 30 °C. At 14°C ascospores infected alfalfa abundantly at 98 %relative humidity (RH) and above, moderately at 97%, sparsely at 95 and 96%, but not at 94% and below. Ascospores were hydrophilic, germinating best at or near 100%, RH but did not germinate at or below 93 % RH. After infection was established, tiny leafspots became visible within 6–7 days at constant temperatures of 15–25°, 10 days of 10°C, 13 days of 5 °C, and 25 days of 2.5 °C. They failed to develop into normal size spots within 4 weeks at constant temperatures near 30 °C, or near 10 °C and lower. Temporary exposure of incipiently diseased plants 1–6 days to 30–38 °C adversely affected subsequent leafspot development at 20–24°C. Inhibition depended on temperature and on the extent of post-infection disease development.     

REVERSIBLE DEACTIVATION OF NEUROSPORA ASCOSPORES BY LOW TEMPERATURE

Sun, Clare Y., and Alfred S. Sussman. (U. Michigan, Ann Arbor.) Reversible deactivation of Neurospora ascospores by low temperature. Amer. Jour. Bot. 47(7): 589-593. Illus. 1960.—Heat-activated ascospores of Neurospora tetrasperma are reversibly deactivated after incubation at 4°C. for 36–48 hr. Two cycles of deactivation and reactivation are possible although the percentage germination decreases in the last cycle. By contrast, spores held at 20°C., or in glycerol at 4°C., will remain activated for much longer periods of time. If an incubation period at 20°C. greater than 30 min. is interposed before the activated spores are placed at 4°C., germination occurs despite the cold-treatment. Furfural-activated ascospores, when held at 4°C., are deactivated but can be reactivated only by heat, pointing up a difference between ascospores activated by these different means. Although a fraction of the stimulus afforded by heat-sensitization to chemical activators is preserved for 2 days at —20°C., it is dissipated completely after a short time at 4°C. These data are discussed on the basis of the suggestion that the reversible production of a substance initiates a series of steps which lead to germination. Thus, the temperature minimum of the forward reaction is greater than 4°C. whereas the back reaction proceeds at this temperature.     

CENTRUM DEVELOPMENT IN DIDYMOSPHAERIA SADASIVANII (PLEOSPORALES)

Development of a typical pseudoparaphysate centrum in Didymosphaeria sadasivanii Ramachandra-Reddy indicates that this ascomycete is properly placed in the Pleosporaceae despite the fact that forcible discharge of ascospores from bitunicate asci has not been demonstrated. The relatively thin-walled asci releasing ascospores within the ascocarp in D. sadasivanii, as in Cochliobolus spp., probably were derived by reduction from the bitunicate type. Ascocarps matured on malt agar slants but developed more rapidly and normally on autoclaved alfalfa stems inoculated in medicine bottles and transferred to moist filter paper in large petri dishes when covered by mycelium.     

High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

White rust of chrysanthemums

Teleutospores of Puccinia horiana Henn. germinate and discharge sporidia between 4 and 23 °C. At the optimum temperature of 17 °C sporidia discharge starts within 3 h. Maximum germination of the sporidia takes place within 2·5 h between o and 30 °C, there being no clear optimum. High humidity and a film of moisture appear to be necessary for germination of both teleutospores and sporidia. Sporidia can penetrate either leaf surface of chrysanthemum to cause infection between 4 and 24 1°C and within the optimum temperature range, 17–24 °C, effectively penetrate within 2 h. The sporidia are very sensitive to desiccation at below 90 % relative humidity. Methods are described, using leaf discs and whole plants, for screening chrysanthemum cultivars for susceptibility to white rust. Cultivars were placed in five classes ranging from susceptible to immune. Leaf discs of immune cultivars can be distinguished within 30 h by a brown discolouration at the point of inoculation. The early stages of development of the fungus in susceptible, resistant and immune hosts are described. The incubation period in susceptible plants is normally 7–10 days, teleutospores being formed a few days later. Leaves become less susceptible with age but the oldest leaves on 5-month-old plants could still be infected. The maximum survival time of teleutospores in the sori on detached leaves was 8 weeks but was considerably less under moist conditions or buried in soil. Low doses of a mancozeb with zineb fungicide controlled infection by preventing penetration rather than by inhibiting sporidial germination.     

Effect of low temperature pre-treatment on infection of clover root hairs by Rhizobium

Summary Holding 24-h seedlings ofTrifolium spp. at 3°C increased the number of root hairs infected byRhizobium when the plants were subsequently inoculated and grown at 19°C. This effect was transitory, disappearing on plants more than 7 days old.     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.     

In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting

Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour × C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.     

Forecasting the airborne spread of Mycosphaerella fijiensis, a cause of black Sigatoka disease on banana: estimations of numbers of perithecia and ascospores

Banana leaves showing different levels of black Sigatoka disease were collected from an unsprayed plantation in Costa Rica during two separate periods representing the wet to dry season transition (October 1993 – February 1994) and the dry to wet season transition (April – September 1995). Laboratory studies were used to investigate the relationship between the release of Mycosphaerella fijiensis ascospores and the amount of inoculum on banana plants showing different levels of infection, as assessed by leaf necrotic area. The number of perithecia present in the necrotic area was used as an indication of potential ascospore loads and was investigated as a series of regression equations. A series of rewetting and incubation regimes was used to investigate spore release under field conditions (21°C and 100% relative humidity in the early morning and 28°C, 60% relative humidity on days when it rained in mid-afternoon). Results suggest that rainfall, combined with a high temperature, may lead to peaks of ascospore release but without necessarily increasing overall numbers released over periods of up to 4 days and that a high level of spore release was less sensitive to changes in temperature once it had been initiated. The exact role of temperature in spore release is still unclear, however, as leaf samples kept at atypically low temperatures also released non-germinating ascospores. An average of 4.5 ascospores was released per perithecium. This does not resolve ambiguities in the literature regarding the number of ascospores present in each perithecium. A linear model relating the average ascospore numbers and necrotic area, using quick estimates of the amounts of necrotic area on the leaves of a random sample of plants across a plantation, is proposed, to give an indication of the relative amount of airborne inoculum potentially available between different plantations.     

The Combined Effects of Rhizobial Nodulation and Nitrogen Fertilization on Growth and Cold Acclimation of Alfalfa (Medicago sativa cv. Saranac)

The combined effects of nitrogen fertilization and rhizobialnodulation on cold-tolerance of alfalfa were investigated. Inoculatedand non-inoculated alfalfa plants were grown under differentnitrogen fertilization regimes equivalent to 0, 25, 50 and 75kg N ha^–1. They were hardened at 2 °C for 4 weeksand submitted to a freezing-test consisting of 16 h at –1°C followed by 3 °C decrement steps. In all nitrogentreatments, inoculated plants grew better than non-inoculatedplants. There was no difference in nitrogen distribution withinthe plants among treatments. Nitrogen levels, but not nodulation,affected the capacity of the plants to harden. However, eventhe plants which were nitrogen-starved hardened, and mean differencesbetween the more- and the less-hardened plants never exceed1.1 °C. Alfalfa, cold-tolerance, hardening, nitrogen fixation, soil nitrogen availability     

Effects of water potential and temperature on the development of eyespot lesions in wheat

Growth of Pseudocercosporella herpotrichoides on potato dextrose agar at water potentials from -0.5 to -6.9 MPa was optimal at 20°C. At 12 and 20°C, six isolates of P. herpotrichoides grew more rapidly at -0.5 to -2 MPa than at -6.9 MPa. Wheat plants inoculated with P. herpotrichoides and grown in columns of soil at either 15 or 20°C developed more severe eyespot lesions under a heavy watering regime than under medium or light watering regimes. P. herpotrichoides penetrated leaf sheaths of inoculated plants grown in compost more rapidly at 10°C night/15°C day temperatures than at 5/10°C; death of leaf sheaths was also more rapid and consequently there were fewer living infected leaf sheaths at 10/15°C than at 5/10°C. Irrigating for 5 wk before harvest increased the severity of eyespot lesions in a 1983 wheat crop.     

Epidemiology and control of powdery mildew (Sphaerotheca pannosa) on roses

In examinations between September 1966 and December 1968 of 741 specimens of rose species and cultivars, cleistocarps of Sphaerotheca pannosa were found on thirty-two cultivars, mostly ramblers and old shrub roses, and on nine of these they were found in two or three successive seasons. On stem pieces placed on soil in each of the winters 1966-7, 1967-8 and 1968-9 the number of cleistocarps with asci and ascospores decreased during November and December and rose slightly in January, but none showed dehiscence. Cleistocarps on rose bushes examined during the winters of 1967-8 and 1968-9 showed a progressive degeneration of ascospores, and by December none was found. Perennation of S. pannosa in buds was demonstrated by field observations, by inducing bursting of dormant buds on surface-sterilized shoots and by dissection of dormant apical buds. In field studies in 1968 of the development of mildew, infected buds were noted on 22 March but secondary infections did not appear until 17 April, though viable conidia and susceptible leaf tissue were present during this period. Low temperatures appeared to be partly responsible for this lag. On detached leaves in the laboratory the fungus developed from germination to sporulation in 4 days at 20°, 7 days at 15°, 11 days at 10° and 28 days at 3°. Keeping inoculated detached leaves at 0° for 10 days apparently did not affect the viability of the conidia. In both 1967 and 1968 there were two host growth periods, each culminating in flowering, between May and September; mildew did not develop on the shoots until the second growth phase, then the disease increased logarithmically on shoots and blooms during August and September. The disease on the shoots was effectively controlled in the field during 1968 by applications of ‘Benlate’ (benomyl) or dinocap, but not by methy-rimol; these fungicides were less effective in controlling mildew on pedicels. Laboratory tests showed that ‘Benlate’ inhibited sporulation of S. pannosa by deforming the conidiophores.     

Movement ofMycosphaerella fijiensis spores and sigatoka disease development on plantain close to an inoculum source

Investigations into the short-distance dispersal of ascospores and conidia ofMycosphaerella fijiensis and subsequent disease development, from point sources of inoculum, were carried out in a small plot of 100 initially uninfected plantain plants in Costa Rica during 1995. Plants were examined every 4 days from the time of planting in early May for the first appearance of disease symptoms. After 3 months, all plants were infected. Levels of inoculum within the plot were monitored with spore traps and weather data were collected. Results suggested that Black Sigatoka disease was spread on the wind, with conidia ofM. fijiensis probably responsible for short-distance dispersal and ascospores for spread of the disease over longer distances. No evidence was found to support splash dispersal of these spores.     

Ultrastructure Changes Induced by Stem Nematodes in Hypocotyl Tissue of Alfalfa

Scarified seeds of Medicago sativa L. ''Ranger'' and ''Lahontan'' alfalfa were allowed to imbibe water for 36 hr and then were inoculated with stem nematodes, Ditylenchus dipsaci Kühn. Seedlings were grown in sterilized Provo sand at 20 C and hypocotyl sections harvested at 1, 3 and 7 days. Evidence from electron micrographs indicated that cells of noninfected control plants contained normally developing chloroplasts bearing stroma, thylakoids, starch grains and plastoglobuli. The cytoplasm contained a nucleus, endoplasmic reticulum, vacuoles, mitochondria, ribosomes and dictyosomes. No morphological symptoms of nematode infection were observed in infected plants of either Ranger of Lahontan alfalfa 1 day after inoculation. Electron micrographs of tissue from the infected plants, however, indicated more osmiophilic bodies (lipid bodies) per cell than did the noninfected control, with more lipid bodies present in Ranger than in Lahontan. Three and 7 days after planting, swollen hypocotyls could be seen; the degree of swelling was greater in Ranger than in Lahontan. Electron micrographs of infected tissues indicated that both cultivars were undergoing the same kind of damage. Injured organelles were endoplasmic reticulum, chloroplasts and the nucleus. Histochemical staining indicated no changes in the middle lamellae.     

Influence of Air Temperature on the Release of Ascospores of Venturia inaequalis

Abstract The influence of air temperature on the release pattern of Venturia inaequalis ascospores was studied by volumetric spore samplers in two spore sampling periods. In the first period (1991–1996; Passo Segni, Ferrara), 15 ascospore dispersal events were considered occurring in daylight, with high spore counts (168–5892 ascospores per m³ air per event), at an average temperature between 8.4 and 20.3°C. Both the length of the ascospore release period and distribution of airborne spores over time were significantly influenced by temperature. A logistic regression model was used to fit the proportion of ascospores trapped from the orchard air as a function of time after the beginning of the discharge event and air temperature. The accuracy of this equation was tested against data collected in the second spore sampling period (1997–2000; Sala Bolognese, Bologna, and Castelfranco, Modena); 16 dispersal events were considered, triggered by rainfall that occurred both in the dark and in daylight, with low to high spore counts (29–458 ascospores per m³ air per event), at an average temperature between 2.8 and 14.3°C. There was a general agreement between the proportion of ascospores trapped from the orchard air during these events and that estimated by using the logistic equation – in most cases, actual and estimated values showed a high coincidence. Statistical comparison showed a significant correlation (r=0.93, P < 0.01) between observed and estimated data.     

Environmental Factors Influencing the Dispersal of Venturia inaequalis Ascospores in the Orchard Air

A 6-year study was carried out in an apple-growing region of North Italy by trapping airborne ascospores of Venturia inaequalis with a volumetric spore trap operated continuously during the ascospore season, with the aim of better defining the weather conditions that allow ascospores both to discharge and to disperse into the orchard air. A total of more than 60 ascospore trapping events occurred. Rain events were the only occurrences allowing ascospores to become airborne (a rain event is a period with measurable rainfall ≥0.2 mm/h – lasting one to several hours, uninterrupted or interrupted by a maximum of two dry hours); on the contrary, dew was always insufficient to allow ascospores to disperse into the air at a measurable rate, in the absence of rain. In some cases, rain events did not cause ascospore dispersal; this occurred when: (i) rain fell within 4–5 h after the beginning of a previous ascospore trapping; (ii) rain fell at night but the leaf litter dried rapidly; (iii) nightly rainfalls were followed by heavy dew deposition that persisted some hours after sunrise. Daytime rain events caused the instantaneous discharge and dispersal of mature ascospores so that they became airborne immediately; for night-time rainfall there was a delay, so that ascospores became airborne during the first 2 h after sunrise. This delay did not always occur, and consequently the ascospore trapping began in the dark, when: (i) the cumulative proportion of ascospores already trapped was greater than 80% of the total season's ascospores; (ii) more than one-third of the total season's ascospores was mature inside pseudothecia and ready to be discharged.     

Effects of environmental conditions on the fixation and transfer of nitrogen from alfalfa to associated timothy

Nitrogen fixation (NF) by alfalfa and nitrogen transfer (NT) from alfalfa to associated timothy was studied under different environmental conditions in controlled growth chambers, using the¹⁵N dilution technique. Evidence was obtained of NT from alfalfa to the associated timothy. Conditions that favored NF by alfalfa resulted in an increase in its NT. Of 3 different temperature regimes (25/20, 16/14, and 12/9°C day/night), 16–25/14–20°C was the best range for NF by alfalfa and resulted in the greatest NT. High light intensity (550 uE.m⁻².sec⁻¹) and long days (16–20 h) also caused increased NF by alfalfa and benefitting timothy more than in a regime of low light intensity (by shading 50% or 75%) or short days (12/12 or 16/8 h day/night). When the inoculated (Rhizobium meliloti) root systems of plants were kept free from other microorganisms (axenic condition) to minimize possible decomposition of dead tissues, lower NT from alfalfa was observed, especially at later cuts, compared to non-axenic plants. This suggests that both direct excretion and decomposition of dead alfalfa tissues are sources of N benefit from alfalfa to associated timothy. Contribution no 1065 of the Plant Research Centre.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Ice tolerance of cold-hardened 'Valencia' orange wood.

Seedlings of Valencia orange were coldhardened in environmental chambers and inoculated with ice at different temperatures during controlled freezes. Hardening resulted in more than a 3 °C decrease in temperature for injury to the wood and a 2 °C decrease for injury to the leaves. The main stems of hardened plants withstood ice without injury for periods three times as long as those for unhardened plants. Hardening also reduced by one-half the rate of linear ice spread in the wood and maintained oxygen uptake activity under conditions metabolically destructive to leaves of unhardened plants.