Fusarium head blight (FHB) is one of the most devastating diseases of small-grain cereals. Severe epidemics have occurred all over the world, affecting wheat in all cropping areas around the world, including those in Argentina, altering the yield and quality of grains, as manifest in their weight, carbohydrate and protein composition, and the mycotoxins presence such as deoxynivalenol (DON) [
Phalip et al. [
Another group of relevant enzymes in phytopathogenic process are those catalyzing proteolysis, referred to as proteases or proteolytic activities. Together with the CWDEs, the proteases act at an early stage of infection to degrade the structural proteins of the cell walls in order to invade the host. At a later stage these enzymes are responsible for the degradation of the grain’s storage proteins, altering the quality parameters of raw material [
Although the role of lipases in the infection process by
Regarding the mycotoxins, it is considered that they may have a more consequential influence on the progress of infections on cereal plants than as phytopathogenicity factors determining the capability of infection [
The high genetic diversity present in
Eleven
Wheat grains were irradiated with 10–12 KGrays of gamma irradiation to retain the grain germinative ability. The grains were checked for sterility and absence of deoxynivalenol (DON) and stored aseptically at 4°C. Flasks were subsequently refrigerated at 4°C for 48 h with periodic manual agitation to allow absorption and equilibrium. Finally,
Rehydrated wheat grains were placed in sterile 9 cm Petri dishes to form a monolayer of grains (20 g). Then a 4 mm diameter agar disk was taken from the margin of a 7-day-old growing colony of each isolate on potato dextrose agar (PDA) at 25°C and transferred to the centre of each plate. Petri plates were placed in closed plastic containers together with beakers of glycerol-water solution at 0.995
DON analyses were carried out following the methodology proposed by Cooney et al. [
The evaporated extract was resuspended in 500
Data were analyzed statistically using PROC GLM in SAS program (SAS Institute Inc., Cary, NC, USA) through an ANOVA. Means were compared by Fishers LSD test to determine the significant difference between the different treatments assayed.
Eleven
Polygalacturonase activity produced by
Protease activity produced by
Lipase activity produced by
No DON was found, as expected, at early stage of infection (3 days) for any of the analyzed isolates. Then, two of them (1 and 2) were able to produce higher concentrations as incubation time increased reaching maximum DON levels at 10 days of incubation, with no statistically significant difference (50.7 and 52.3
Mean DON production (
The analysis of variance of the effect of single factor (isolates and days) showed that all factors alone and all interactions were statistically significant (
Analysis of variance of days (
Variation source | df | PGase | Protease | Lipase | df | DON | ||||
---|---|---|---|---|---|---|---|---|---|---|
MS |
|
MS |
|
MS |
|
MS |
| |||
|
10 | 23264.22 | 174.31* | 332.57 | 872.37* | 767.70 | 1923.79* | 3 | 1479.13 | 190.63* |
|
14 | 12725.49 | 95.35* | 238.63 | 625.97* | 389.02 | 974.87* | 4 | 4414.26 | 568.92* |
|
140 | 824.42 | 6.18* | 11.90 | 31.22* | 34.38 | 86.15* | 12 | 371.73 | 47.91* |
|
0.77 | 0.95 | 0.97 | 0.99 |
df: degrees of freedom.
MS: mean square.
*Significant
Since the substantial economic losses in cereal result from
FHB infection consists of two phases, initial infection and spread of disease symptoms within a wheat spike. In the first 48 hours the initial biotrophic phase develops, with growth of intercellular fungal hyphae in the host, being the stage where enzymes play a decisive role. Then, the necrotrophic phase is developed with intracellular hyphae growth in the host and beginning of mycotoxin production [
The infection process of
The present study provides tools as selection criteria of
Since the aggressiveness is determined by various factors and variables, different criteria can be used to estimate the infectivity of the inoculum obtained from isolates. The criteria used are estimates, not conclusive, which could be considered as complementary data. For this reason, the detection and analysis of some enzymatic activities were selected, according to the relevance of their function in the infective process. The pectinases are crucial to start the infection process, allowing the action of other enzymes. On the other hand, the loss of grain quality for their marketing focuses on the action of proteases on storage proteins and mycotoxin content. Moreover, although lipases have been scarcely studied, they would act to a certain extenst in the prior degradation of the external cuticle. Since this paper analyzes simultaneously both, the
In this report, the PG and proteolytic enzymatic activities were detected for all isolates in an early stage of the incubation time. Regarding the lipase activity, only in two isolates the activity was not detected, reaching in the other isolates the maximum value at the higher incubation periods. In general, production patterns obtained during the incubation time were similar, with a different magnitude.
In fact,
Our results showed that at least a minimum of five days of incubation were required for some isolates (and even more incubation time for the others) to produce detectable DON concentrations, which would infer its relation with the progress of the disease in wheat, more than with the early stages of infection by
Based on antecedents, it is noted that the role of mycotoxins in plant disease has been controversial. In FHB disease, there are diverse interactions between wheat genotypes and pathogen isolates, which makes it difficult to understand completely the role of DON in the pathogenesis. The different mechanisms of resistance of wheat would be in relation to the difficulty in interpreting DON concentrations detected in the infection [
Jansen et al. [
Although the results obtained determined that numerous isolates had high enzymatic activities related to infection process, what is remarkable is the isolate number 1 for its production, which had also high toxicogenic capacity, so it could be selected for further research on the evaluation of the disease on different wheat genotypes.
Since FHB is one of the most devastating diseases on wheat that alter the yield and quality of the grain worldwide, an earlier characterization of
There is not any kind of conflict of interests with any trademark mentioned in this paper, competitive interest, or secondary interest that could have influenced the research. This declaration is carried out by all the authors of the work presented.
The authors thank Consejo Nacional de Investigaciones Científicas y Tecnológicas (Grant PIP 1422) and FONCYT PICT 2011-0851 for financial support and acknowledge the technical assistance of Bernardina Catalina López and Esteban Manuel González.