Dithianon is a fungicide commercialized since 1963 with a broad spectrum of activity on many phytopathogen fungi belonging to Ascomycetes or Deuteromycetes  that infect crops all over the world  and is registered on many diseases, comprehending also on Venturia (inaequalis and pirina).
It is a multi-site fungicide and its mode of action, not well known, is based on activity against thiol groups in proteins and peptides, especially glutathione  . It is substantially a surface product with preventive applications.
In the control of V. inaequalis, dithianon has often acquired an important role and it is active mostly in preventive applications, 3 - 5 day before infection  , but also in timely post-infection sprays, better 12 hours after the beginning of the infection  . It is utilized during a long vegetative period (from “mouse ears” to “fruit set”), generally with a rather high number of treatments. The importance of this fungicide is increased by the fact that regulations of European Community  revoked or limited many pesticides, reducing particularly those with multiside action and favoring the news, more toxicologically sure but with a much higher risk of resistance.
In bibliography, there are not reports about loss of sensitivity and activity caused by reduced sensitivity, also because dithianon is generally used against diseases that do not require a high number of treatments or it is alternate with other fungicides.
In some Italian apple areas the number of treatments with dithianon increased, over 10 or 15, caused by the occurrence of resistance to other fungicides, as QoI’s , AP’s  and DMI’s .
Subsequently to this context, the technics of some Italian apple areas had the suspect that the dithianon could present a reduced activity.
Therefore, we set up researches to assay different methodologies to verify if these reductions could be possible.
Three types of Venturia inaequalis populations with different origin and scab management (and with presumably different sensitivity) were collected from apple areas and assayed with different methodologies.
The different types were collected from:
- Trees in uncultivated and untreated areas (sensible-wild-types).
- Orchards often treated with dithianon where the control of V.inaequalis was good. There was also a population “mixed” constituted by a mix of conidia from many “well (good) controlled” populations of several Italian apple areas (sensible, treated).
- Orchards often treated with dithianon, with control complains and suspects of reduced activity by this fungicide (“poorly controlled” and suspects on dithianon).
The fundamental comparison was between well and the poorly controlled population with suspects on dithianon. The methodologies assayed were:
2.1. In Vitro
The dithianon was tested as antigerminative activity on conidia of V. inaequalis populations in Petri plates amended with the fungicide  utilized as active material (PESTANAL® Sigma-Aldrich International). Every sample was tested with two or three replicates. In each one, a part of inoculum (subpopulation) constituted by about 0.014 g of scabbed leaves was shacked in sterile water, filtered and adjusted to a conidia concentration of 1 - 25/ml. Drops of 20 µl from this dispersion were put in agar with the following concentrations of dithianon: 0, 0.01, 0.1, 0.3, 1, 3, 10, 30 mg/L. Before dilutions, streptomycin sulphate (200 mg/L) was added. The percentage of germination was assessed 24 hours later and data transformed in probits to obtain EC50 values (the concentration reducing of 50% the spore germination respect the test). The data were transformed to obtain RF (Resistance Factor: mean EC50 each treated population/mean EC50 sensible ones).
The same germination data were used also for assessment of M.I.C. (Minimal Inhibiting Concentration able to completely inhibit germination of spores) and concentrations used were: 0.3, 1.0, 3.0, 10 and 30 mg/L. Another used parameter is the RF (Resistance Factor) as the ratio: EC50 examined population/EC50 reference sensible population.
The data were collected and examined in 2008 and 2010 year, and compared with sensible ones.
The EC50 values were analyzed with a model of variance with years and populations as effects (Statgraphics 12.0, P ≤ 0.05).
These tests were realized with different methodologies on different types of apple plants:
2.2.1. Apple Seedlings
Apple seedlings cv. Golden with 5 - 6 leaves were preventively or curatively sprayed with dithianon. Different concentrations: 0.25, 0.50, 1 (field dose) and 2 g/L as commercial formulate of Delan (70 WG, 70% a.m., BASF) were used in preventive treatments. In curative ones only the field dose (1 g/L) was used
In preventive ones, 24 hours after applications, the plants were inoculated with a suspension of conidia by populations of V. inaequalis with a concentration of 1 - 2 × 105 conidia. After 48 hours of incubation with continuous wetness and 20˚C as mean temperature, the leaves of seedlings dried up and the curative application was realized. Subsequently all plants were incubated for 20 - 21 days at about the same temperature. In the end, the assessment of infection was carried out as mean percentage of scabbed leaf area and relative efficacy (%)*. The control, in field and in vivo trials, is classified as “good” when efficiency % is superior to 90% and “poor” when it is inferior. Statistical elaboration on grafted apples will give a more precise value of “good or poor control” in thesetrials.
2.2.2. Grafted Apple Plants
After the two previous assays, a new type of in vivo trials (for dithianon) was by me pointed out and realized on potted grafted apples cv. Imperatore Dallago (very sensitive to scab, comparable to cv Mc Intosh), two or three years old, with 4 - 6 branches.
The two groups of populations were compared: sensible (well controlled ones) confronted with the poorly controlled ones with suspects on dithanon, while wild-types were not utilized, considering the high amount of work and being less essential for the comparison respect the last two types. The sensible ones were represented by a population (n.608) well controlled in Ravenna province and another sensible defined “mixed” constituted by conidia of several populations (already declared “In Vitro tests”).
In comparison, populations from Emilia-Romagna Region and Trentino Province with suspects of reduced control by dithianon were tested (poorly controlled).
From two to four replications were carried out for every population. In each test and population, four plants were inoculated: two as control ad two treated with dithianon. The application was realized 16 hours before the inoculation and every plant was treated with about 35 ml of dithianon as commercial product Delan 70 WG (70% active material), BASF using 1 g/L. The last formed leaf of every bud was detected with a plastic bind and application was realized with a handle mechanic sprayer, until the drip limit.
Inoculations were realized utilizing, for each plant, about 0.007 g of scabbed leaves and 14 ml of distilled water. The amount of scabbed leaves and water was soaked, filtered and the conidia concentrated 0.8 - 1.35 /ml.
They were maintained in glasshouse with a temperature about 20˚C and relative humidity 60% - 98%, while the assessment of infection was realized 21 - 24 days after inoculation on 30 - 60 leaves/plant, evaluating the percentage of scabbed leaf area. The data were transformed in relative efficacy %*.
2.2.3. Statistic Analysis
The relative efficacy of dithianon was calculated starting from the mean percentage of scabbed area on leaves of the group of sensible and well-controlled populations, compared with that of suspected ones. Activity data, expressed as efficacy (%), were subjected to Bliss transformation (arcsen√efficacy %) and elaborated as “incomplete and unbalanced randomized blocks” design (SAS 9.0). This program was used for the different number of tests for every sample and, The C.I. (Confidence Interval) on efficacy (%) of sensible and well controlled populations was used to identify a suspected population, as statistically different (Table 4).
3.1. In Vitro Tests
Each population tested in 2008 and 2010 is presented in Table 1 with type of scab management, location, treatments, EC50 and MIC.
The statistic elaboration of EC50 values (Table 2), expressed as mean in each type of population sensitivity, shows that wild-type populations present sensitivity data like that of well-controlled ones but are at the limit of significance with referring to the poorly controlled ones. On the contrary the well-controlled populations present no significant differences with respect to the poorly controlled ones.
3.1.1.In Vivo Tests on Seedlings
Table 3 reports the results referred to the degree of infection and to the efficacy (%) of dithianon applied on several populations inoculated both preventively (with different doses) and curatively (only field dose).
The data show low or moderate levels of activity by dithianon, not only in reduced doses (until 1/4 of field dose), but also at field dose on both the types of tested populations (well or poorly controlled). Increasing the dose (from 1/4 to 2x), the degree of infection generally does not show reductions and consequently the efficacy % does not increase. The same tests replicated on population n. 99 (another well controlled population) show very uneven results, irrespective of each concentration. Likewise curative applications offer moderate or low levels of activity on well or poor controlled populations.
Table 1. Results in vitro sensitivity to dithianon (EC50 & CMI) by populations from wild types, well-controlled and poorly controlled with suspects on dithianon in 2008 and 2010 survey.
* Concentration that reduced germination by 50% respect test; ** Concentration Minimum Inhibiting; *** Referred to the last two or three years; **** Not Detected.
Table 2. Mean sensitivity In vitro tests (EC50 values) of the three types of populations and results of statistic elaboration.
* Values statistically non-significant; ** Values statistically significant; *** Resistance Factor respect wild-types.
Table 3. Results of the degree of infection and the efficacy (%) of dithianon on well and poor controlled populations of V. inaequalis inoculated on seedlings with curative and preventive applications.
* Mean percentage of scabbed area on apple leaves; ** Efficacy %; *** Field dose dithianon: 1 g/L; **** “Good control or well controlled” when efficacy % is superior to 90%; “poor control” when it is inferior and with suspects on dithianon.
3.1.2. In Vivo Tests on Grafted Apple Plants
Table 4 presents each type population, populations, their location and results (relative efficiency %) obtained in each subpopulation. It is visible that there are moderate differences among efficacy % of all populations, both sensible and “suspected of reduced control”.
Table 5 presents statistic elaboration where C are sensible and well controlled populations, while N are those “Suspected of reduced control” and RC all the populations. General statistic diversity presents P > 0.019, so among all populations there are not statistical differences.
We chose the C as referring group, calculating mean percentage. The results expressed as relative efficacy show that the highest levels in assays are presented by good-controlled populations (n. 608 and “mixed”) with values from 100 to 93.3%, with a mean as 96.9% and a confidence interval of 10.6. So subpopulations inferior to an efficacy of 86.6% are considered statistically different.
Suspected samples (Table 4) from Emilia-Romagna region show that five populations (n.704, 603, Ba3, 2915, 601) present non significant values of dithianon efficacy (%) in all subpopulations.
The other four populations (n.501, 602, 615 and 3015) show a part of trials where subpopulations evidence significant reductions of activity by this fungicide, with values inferior to the I.C. (Confidence Interval). In Trentino Province (Table 4) only populations n. 16, 21, 23, 24 present one assay with significantly inferior values. The others, n. 17, 19 and 25 present no significant values of all subpopulations
Table 4. Efficiency (%) of dithianon in vivo (grafted plants) on two types of V. inaequalis populations: Well controlled and suspected of reduced control by the fungicide in two apple areas.
* Reduction of efficacy (%) out of the Limits of Confidence, so statistically significant.
Table 5. Statistic analysis on results of efficacy % of dithianon in test on grafted apple trees.
These reductions are generally light (efficacy percentage superior to 80%) and only two subpopulations (one from sample n. 501 and one from n. 615) present efficacy values inferior to 80%.
With referring to samples from Trentino Province, three populations (n.17, 19 and 25) respect seven total, show light reductions of activity, that are inside “confidence interval”. On the contrary four populations (n. 16, 21, 23, 24) present, in one of the two realized tests, a subpopulation with efficiency values inferior to C.I. of well controlled ones. Nevertheless, the reduction of activity percentage is moderate, with relative efficiency always superior to 80%.
4. Discussion and Conclusions
The research on eventual reductions of sensitivity and activity of V. inaequalis populations to dithianon has been carried out with different methodologies: in vitro as antigerminative activity of dithianon on conidia; in vivo, verifying preventive and curative activity of this fungicide on scab presence of different kinds of plant: seedlings or grafted plants.
In vitro tests, the three types of populations examined (wild type, well-controlled and poorly-controlled with suspects on dithianon) were compared as EC50 and MIC values. The results showed that there are no statistical differences among the three types of populations, especially when we compare well and poorly controlled ones in commercial orchards. The statistical analysis confirmed what was observed by Stammler  with several samples from Germany.
The second methodology, based on preventive and curative applications of dithianon on apple seedlings, was realized with only a few samples of compared populations (well controlled and poorly ones, dithianon suspected), because it was clear that this methodology is not apt to show sensitivity and activity differences, as informed by Stammler (pers. comm.).
It is not clear why the activity of dithianon on apple seedlings was so low, considering that similar tests with seedlings on other fungicides as AP’s  and DMI’s  permit high correlation between this type of test and different sensitivities with reliable results, as shown by me. It was also advised by FRAC .
The last methodology was carried out with populations inoculated on apple leaves, not on seedling but on grafted apple trees and tested as comparison between sensible well-controlled populations and suspected of reduced control. All populations present no significant differences. With referring to subpopulations there are inferior activities in some populations both in Emilia-Romagna and Trentino.
I can detect that a reduction of activity is possible, also for fungicides with multi-site action, as dithianon, but now we examine and compare characteristics.
It must be pointed out that the detected reductions of dithianon in activity appear light and highly inferior to the decreases of sensitivity/activity ascertained by me with other fungicides, as QoI’s  and DMI’s  in presence of resistance by V. inaequalis.
This decline of dithianon is caused by new resistant genotypes, or it is more adequate to impute this to temporary modifications activity and sensitivity by apple scab (V. inaequalis).
In this regard I contacted technics of Emilia-Romagna region and Trentino province, and learned that in the following years these reductions of activity were almost absent, while continuing to use dithianon. This leads to belief that the moderate reductions of sensitivity and activity in surveyed years were determined by temporary modifications in the target of fungicide with biochemical and physiological interactions. They were probably caused by very high infective pressures (meteorological conditions) with difficulties to intervene in time. In addition must be considered the diffused presence of apple cultivars with medium or high susceptibility (initial or acquired) to scab . In very case, an annual number of dithianon treatments not superior to 9 are advised.
Thanks to Ernesto Muzzi for statistic elaboration, Marina Collina for logistic support, the Emilia-Romagna Technical Service for support with samples of this Region, Gastone Dallago and Technic Service S.Michele all’Adige for support in Trentino Province; Angela Finestrelli and Giulia Toschi for support, respectively, in laboratory and greenhouse tests.
Studies financed from Emilia-Romagna Region as part of PSR 2014-2020 Op 16.1.01 GO PEI-Agri-FA 48, Pr. “Resistances” with coordination of CRPV.
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