In vitro production of conidia of Alternaria solani. Sporulation of Alternaria solani can be scarce is often reduced when the fungus is cultivated in vitro. A series of experiments were conducted to assess the effects of moisture, mycelial wounding, light quality and photoperiod, and culture media on conidial production. A procedure to induce sporulation based on mycelial wounding and dehydration was adapted and validated.
The procedure was validated with 30 isolates of A. Conidial production, germination, and infectivity were quantified. Inoculations of conidial suspension of all isolates resulted in lesions tomato and potato plants.
The effect of subculturing on A. After six subcultures, seven days, there was no reduction in sporulation of colonies that were induced with the protocol. Validou-se o protocolo para 30 isolados de A. In vitro experiments are routinely used to study the nature and control of plant diseases caused by fungal pathogens. Sporulation is a key component for several purposes because fungal spores are frequently used as propagules to infect plants.
In the case of potato Solanum tuberosum L. It is well known that in sporulation of A. Three major factors that influence sporulation in vitro are commonly manipulated: For instance, the V8 juice medium, PDA potato-dextrose-agar Asexual reproduction fungi conidial suspension Asexual reproduction fungi conidial suspension with parts or extracts of plants are used in protocols to induce sporulation of Alternaria spp.
Light and temperature affect many aspects of fungal development, including the formation of reproductive structures. In vitro sporulation of A. In Neurospora crassa there are
Asexual reproduction fungi conidial suspension least five photoreceptor groups, which are related to many
Asexual reproduction fungi conidial suspension of the fungus life cycle, including sporulation Purschwitza et al. The factors described above and stress conditions can be properly combined to induce fungal sporulation.
Mycelial injury wounding and gradual loss of the culture media moisture content lead to stress conditions that stimulate sporulation in Alternaria spp. In pioneering work, Walker described the biphasic technique for massive production of conidia of A. The technique is called biphasic because initially the mycelium is produced in liquid medium, wounded, and then transferred to a solid medium to allow sporulation.
Little information is available about the mechanism that triggers the production of reproductive structures induced by dehydration and wounding. Although many studies were conducted to investigate sporulation of A.
Another problem related to these studies is the low number Asexual reproduction fungi conidial suspension isolates assessed. Usually, only a single isolate is used to determine the favorable conditions to allow in vitro sporulation. This can be an important issue for highly variable species such as A.
Therefore, the objective of the present study was to adapt the protocol developed by Walker to induce sporulation in A. Additionally, Asexual reproduction fungi conidial suspension protocol developed was validated with several isolates of the pathogen.
Isolates, cultural conditions, and conidia quantification. Thirty different isolates of A.
Isolates preserved on filter paper were used in all tests to adapt the protocol and to assess the effect of subculturing on conidial production. Isolates preserved under different methods were used.
Ten mL of the mycelial suspension was deposited in 15 mL of culture medium, which varied according to the experiment, in 9 cm-diameter Petri plates.
Three days after the beginning of the experiments, 10 mL of DW plus 0. The apparatus used to provide light during
Asexual reproduction fungi conidial suspension incubation of the colonies had six light-bulbs spaced 5 cm apart and located 30 cm above the plates. Effects of culture medium dehydration and mycelium wounding on sporulation. Three treatments were compared: Two isolates AS and AS were used in the experiment to quantify the effect of culture medium dehydration on sporulation.
Plates without lids were kept under black and daylight bulbs, as described above. Every 12 h the plates were weighed, until 84 h after the beginning of the experiment. All subsequent experiments used mycelium wounding and plates without lids, to allow for dehydration and fungal sporulation.
Effects of the source of light, photoperiod, and culture medium on sporulation. Three sources of light Asexual reproduction fungi conidial suspension compared: In this last treatment, the bulbs were alternated. Each source was tested a continuous light regime or in a 12 h photoperiod. Three plates with V8 agar-CaCO 3 medium per isolate, per treatment combination of light source and regime were used.
Plates kept under continuous darkness were used as controls. Four culture media, amended or not with CaCO 3, were compared: The pH was adjusted to 7. Asexual reproduction fungi conidial suspension on the results
Asexual reproduction fungi conidial suspension the previous experiment, the plates were under black light, 12 h photoperiod.
Three plates were used per isolate, per treatment. Effect of periodic subcultures on sporulation. Three plates and three flasks were prepared per isolate at each subculturing. Every 7 days, cultures in plates were subcultured. After colony development, the cultures in liquid medium were Asexual reproduction fungi conidial suspension and induced to sporulate using the best combination from the previous experiments.
Validation of the protocol to induce sporulation and infection. Two experiments, the first with Asexual reproduction fungi conidial suspension isolates and the second with 30 isolates, were undertaken to validate the protocol.
Conidial production was induced in five plates per isolate. Three slides were prepared per isolate. In the second experiment, the incubation period was 6 h. A conidium was considered as germinated when one germ tube was at
Asexual reproduction fungi conidial suspension as long as the conidium' s width. One hundred randomly selected conidia were examined per slide. To assess the infectivity of the spores, 45 to day-old tomato plants cv. Santa Clara or detached leaflets of potato cv.
Detached leaflets of potato were used because the number of plants was not enough to conduct the experiment.
Each tomato plant was divided into three thirds upper, middle, and lower based on plant height. In each third, three leaflets were tagged with a paper tag and a cover glass 1 x 1 cm was
Asexual reproduction fungi conidial suspension on the adaxial surface with double-faced adhesive tape.
The conidial suspension was sprayed on the leaflets using a DeVilbiss 15 sprayer. Nine detached leaflets of potato were inoculated as above, and kept in a plastic box. After the inoculation, the cover glass was removed and placed upside down on top of a drop of lactophenol on a microscope slide. The number of conidia deposited on the cover glass was counted under the microscope X. The number of lesions formed in the leaflets was counted on the third and fourth day after inoculation.
Based on the results of experiment 1, only leaflets of the middle third of tomato or potato plants were used in experiment 2. The infection frequency IFconsidered as the ratio between the number of lesions L and the number of conidia deposited on a leaflet, was used to assess infectivity.
Leaflet area was measured
Asexual reproduction fungi conidial suspension the fifth day after inoculation, using an electronic leaf area meter Li The cover glass area 1 cm 2 was subtracted from the total leaflet area to result in a corrected number of spores deposited on a given leaflet CN. The IF was determined as: A two-way analysis of variance was conducted to analyze the data.
Whenever the treatment x isolate interaction term was significant, the analyses of the Asexual reproduction fungi conidial suspension effects were done by Each experiment was conducted twice hereafter referred to as Asexual reproduction fungi conidial suspension 1 and 2except those for the quantification of culture medium dehydration, infection frequency, and subculturing.
The Levene test was used to check the homogeneity of variance between assays conducted twice Kuehl, In the experiment with culture media, the pH treatment effect was evaluated by contrasts. Data from each assay were analyzed separately because of the lack of homocedasticity. Aerial mycelium growth with no sporulation was observed in colonies formed in plates with lids, sealed or not with Parafilm. In open plates, medium dehydration occurred and vegetative mycelia were sparse, and produced asexual structures.
The sporulation varied among isolates, and ranged from 3. After 60 h, no loss of moisture was observed until the last observation at 84 h. Data from both experiments were pooled and analyzed. Asexual reproduction fungi conidial suspension isolates sporulated under continuous darkness.
The data from the assays of culture media were analyzed separately. The interaction between culture medium supplemented or not with CaCO 3 x pH was significant in three of 16 interactions in assay 1, and in 2 of Asexual reproduction fungi conidial suspension interactions in assay 2 Table 2. Every colony sporulated until the 6 th subculture. Different colonies of the same isolate did not differ regarding sporulation.
At least one colony of each isolate sporulated until the 9 th
Asexual reproduction fungi conidial suspension. For AS and AS, at least one colony produced conidia until the 18 th subculture and one colony of the isolate Asexual reproduction fungi conidial suspension sporulated until the 20 th subculture Figure 2. The following protocol was defined: All isolates were pathogenic to their original host species, but the infection frequency varied.
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In vitro play of conidia of Alternaria solani. Sporulation of Alternaria solani can be at a premium and is regularly reduced when the fungus is refined in vitro.
A series of experiments were conducted to assess the effects of moisture, mycelial wounding, candle grandeur and photoperiod, and enlightenment media on conidial motion. A methodology to create sporulation based on mycelial wounding and dehydration was adapted and validated. The means was validated with 30 isolates of A.
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Not what I thought ??Inoculations of conidial suspension of all isolates resulted in lesions on tomato by using unfavorable conditions for vegetative growth (Rotem & Bashi, ; Shahin of fungal development, including the formation of reproductive structures. conditions, affecting asexual reproduction of these fungi as individual mycelia rated flasks, conidia are often kept in suspension for up to several days, and a..
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Every 12 h the plates were weighed, until 84 h after the beginning of the experiment. Services on Demand Journal. In Neurospora crassa there are at least five photoreceptor groups, which are related to many aspects of the fungus life cycle, including sporulation Purschwitza et al. Vieira BS Alternaria euphorbiicola como micoherbicida para leiteiro Euphorbia heterophylla: In the present work, greater sporulation was obtained under alternated regime of light 12 h photoperiod. In the second experiment, the incubation period was 6 h.
All subsequent experiments used mycelium wounding and plates without lids, to allow for dehydration and fungal sporulation.
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