In vitro relationship between dithiocarbamate residue and Stemphylium vesicarium infection on pear fruit

Crop froreckm Vol. 14, No. 4. pp. 321-326. 1995 Copyright @ IYYS Elsevier Science Ltd Printed m Great Rritain. All nghts reserved OXI-2194% $10.00 + O.00

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In vitro relationship between dithiocarbamate residue and Steeq&yI__~~ vesicarium infection on pear fruit A. Marchi,* A. Folchi, G. C. Pratella and D. Caccioni CRIOE University of Bologna, via Filippo Re 8, 40121 Bologna, Italy

Abate F&l pear fruits were collected at three different stages of development and treated with one of two dithiocarbamates (thiram or metiram) at concentrations ranging from 0.4’to 3.2 g I-‘. The residue expressed as carbon sulphide (CS2) was then measured using spectrophotometry and correlated with fungicide dose and with development stage. Generally thiram was found to leave higher residues on the fruits than metiram. For each product, at equal doses of active ingredient, the amount of residue decreased as sampling date approached harvest. This pattern could not be attributed to a change in the surface:volume ratio since there was no significant fruit size growth between the second and third sampling dates. Fungitoxicity tests showed that thiram was more effective than metiram against conidial germination, Stemphylium vesicarium with respective EC 50 values of 2.3 mg kg-’ and 14 mg kg-‘. Thiram was also found to be more effective than metiram in preventing Stemphylium vesicarium infection on pears. The relationship between disease severity and amount of residue (CS2) left on the fruit after treatment was found to be linear for metiram and exponential for thiram. However the residue resulting from the fungicide dose recommended for field treatment (0.7-0.8 g I-’ active ingredient), corresponding to a CS2 residue of less than 1 mg kg-‘, did not guarantee effective pear protection under the conditions tested. The efficacy of this treatment in the field was related to residue accumulation following repeated treatments, or to conditions less conducive to the disease onset than those tested in the laboratory.

Keywords: Stemphylium vesicarium; pear; Pyrus communis; dithiocarbamate; fungicide residue

Stemphylium vesicarium (Wallr.) E. Simmons is a demaziaceous hyphomycete (Deuteromycotina) mainly known as the cause of blight to onion (Allium cepa L.) (Miller, Taber and Amador, 1978; Shishkoff and Lorbeer, 1989), asparagus (Asparagus officinalis L.) (Blancard, Piquemal and Gindrat, 1984; Gindrat, Varady and Neury, 1984; Falloon, Falloon and Grogan, 1987) and, to a lesser extent, of tomato blight (Lycopersicon esculentum Mill.) (Porta-Puglia, 1981). Recently S. vesicarium has been found to be responsible for a serious disease attacking pears (Pyrus communis L.) in Italy (Ponti, Cavanni and Brunelh, 1982; Ponti and Cavanni, 1983), in France (Blancard, Allard and Brest, 1989; Cugier and Humbert, 1991) and in Spain (Vilardell, 1988; Montesinos and Vilardell, 1992). The early symptoms of the disease usually affect the leaves and, in rarer cases, the twigs. The most serious damage, however, occurs on the fruit and, in some cases, the attack is not preceded by any infection of other plant tissues. The pathogenicity of S. vesicarium is associated with an unknown host-specific toxin that necrotizes the epidermal tissue, favouring colonization by the fungal mycelium (Ponti and Cavanni, 1983). Fruit loss as a consequence of this disease in the pear

*Author to whom correspondence

should be addressed.

vary from 1% to as much as 90%. The most serious losses to cultivars of this crop have been reported by growers in the PO Valley area. Among the cultivars affected, the one that appears to be the most sensitive is ‘Abate F&e1 (Ponti et al., 1982). On the basis of current knowledge, the best method for reducing disease loss caused by S. vesicarium is through several applications of dithiocarbamate fungicides, to ensure that all susceptible plant organs are thoroughly protected at all times (Ponti et al., 1993). Treatments normally cover the period from end of bloom to harvest and are carried out every 6-8 days. Frequent treatments may, however, give rise to residues higher than those permitted by law, which according to current Italian (Ordinanza Ministeriale 3 maggio 1994, Gazzetta Ufficiale della Repubblica Italiana, n. 160, Roma 11 luglio 1994), Spanish (Real Decreto 28011994, de 18 de febrero, Boletin Oficial de1 Estado, suplemento n. 68, 9 de Marzo de 1994), and French (Arrete du 5 aotit 1992, Journal Officiel de la Republique Francaise, 22 septembre 1992) regulations must not be greater than 2, 3 and 1 mg kg-’ carbon sulphide (CS,), respectively. The purpose of this study was to investigate, through laboratory testing, the relationship between S. vesicarium infection and dithiocarbamate residue, to determine the minimum amount of residue necessary for adequate fruit protection. may

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Materials and methods Source of fruit and post-harvest fungicide treatments Abate Fete1 fruits from an orchard in full production were collected at three different stages of development: 38, 17 and 2 days, respectively, before commercial harvest. Fruits were treated in the orchard with ziram at a concentration of 1.36 g 1 a.i. at intervals of 8-10 days. Altogether, fruit collected at the first sampling date received 12 sprayings of this type in the field, three more sprays were applied before the second date, and there were no further treatments before the third. A subsample of 30 fruits were selected for each date in order to determine their weight, size and height. In order to lower dithiocarbamate residues from field treatments, fruits were first washed in a 6% sodium silicate solution for 30 s and then rinsed under running water for 5 min so as to eliminate all traces of the salt (Pratella, Folchi and Macaferri, 1989). After thorough drying, fruits were carefully sampled and treated by dipping for 20 s in metiram (PolyramCombi, BASF-ITALIA) or thiram (Silfur GD 50, SIAPA) with the exception of the second date, for which thiram alone was employed. Rates of both fungicides tested were 0.4, 0.8, 1.6, or 3.2 g a.i. 1-l. Control fruits were dipped in fresh water. There were 60 fruits per treatment, divided into five replications with 12 fruits each. Two fruits. were taken from each replication, so that the residue was measured on a total of 10 fruits for each treatment. The rest of the fruits were used in inoculation tests.

Residue determination Residue analyses were carried out by the calorimetric method in order to determine the amount of carbon sulphide (CS,) which is formed from dithiocarbamate residue by acid hydrolysis in the presence of concentrated hydrochloric acid, stannous chloride and potassium iodide. The carbon sulphide formed was absorbed by an ethanol solution containing copper acetate and diethanolamine, thus giving rise to a yellow chelate [copper salt of N,N-bis-(2-hydroxyethyl)-dithiocarbamic acid], which was measured by means of a spectrophotometer at a wavelength of 435 nm (Gordon, Shuckert and Bornak, 1967; Keppel, 1971). The residue (expressed as CS,) was measured four times, on each set of 10 fruits, for each treatment. For each sampling date, dithiocarbamate residue was measured four times, before and after washing in silicate solution, on a set of 10 fruits. Inoculation test The strain of S. vesicarium used throughout the tests was isolated from infected pear and cultured on malt agar for 1 week at 25°C under ultraviolet light with a 12 h light/dark cycle to induce sporulation. The conidial suspension, obtained by washing the colonies with a 0.05% Tween 80 solution, was adjusted to a concentration of lo5 conidia ml I-’ and sprayed onto the fruits placed on a rotary table through two nozzles set at 81 KPa for 1.5 min. After treatment, the fruits were placed in trays wrapped in polyethylene bags and

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periodically sprayed with water in order to maintain high relative humidity values. After 17 days in stbrage at 2O”C, the disease severity was evaluated on the basis of five classes where 0 was no symptom, 1 was few lesions less than 2-3 mm in diameter, 2 was many lesions less than 2-3 mm in diameter or few lesions with larger diameter, 3 was many lesions all over the fruit coalescing at some point, and 4 was lesions converging with wide necrotic areas covering about one-third of fruit surface. Recovery of the pathogen from inoculated fruits After inoculation on the rotary bench a sample of five fruits taken from the test was allowed to dry in a laminar flow hood. Each fruit was separately immersed in a beaker containing 200 ml of sterile water (Tween 80, 0.05%) and then shaken on a rotary shaker at 60 rpm for 20 min. To improve detachment of the microflora from the carposphere (Martini, Federici and Rosini, 1980) the sample was sonicated (Transonic TS 540, ELMA, D-7700 Singen/Htw, Germany) for 20 s and then further shaken for 20 min. The washing water was then filtrated through a cellulose nitrate filter of 5 urn pore size and the residue left on the filter was resuspended in 4.5 ml of sterile water (0.05% Tween 80) by thorough mixing using a vortex. The number of S. vesicarium propagules was assessed following the Plate-Dilution Frequency Technique described by Harris and Sommers (1968). A 4-fold dilution series was plated in three replicates on dichloran chloramphenicol peptone agar (DCPA), a selective medium for dematiaceous hyphomycetes (Hocking and Andrews, 1987) modified as follows: bacteriological peptone, 15 g; K2HP04, 1 g; MgS04*7Hz0, 0.5 g; chloramphenicol, 0.1 g; dichloran (0.1% solution in ethanol), 1 ml (equivalent to 1 mg 1-l); agar, 20 g; distilled water 1 1. Plates were checked for S. vesicarium growth after 1 week incubation at 25°C under ultraviolet light with a 12 h light/dark cycle. The results were expressed as number of S. vesicarium propagules per fruit. Fungitoxicity test Fungicides were dissolved in acetone and suitable dilutions were added to potato-dextrose agar (PDA) cooled at 45-50°C. For each fungicide four concentrations ranging from 1.8 to 48.6 mg 1-l a.i. were prepared in geometric progression. The solvent concentration in both test and amended PDA never exceeded 1%. After thorough mixing, the agar was poured into Petri dishes in 20 ml aliquots and four plates per thesis were seeded each with 0.2 ml of a pathogen conidial suspension adjusted to 3 X lo5 conidia/ml. The suspension was spread on the agar surface with a bent glass rod and after 24 h incubation at 25°C 100 spores per replicate were examined for germination. To facilitate germinated spore detection a few drops of 0.1% Lacto-fuchsin were added to the agar surface. Statistical analysis Statistical analyses were done with the SYSTAT 5.2 (Systat, Inc., Evanston, 11, U.S.A.) software package. The effect of sampling date on fruit growth parameters,

Dithiocarbamate

residue and S. vesicarium

infection: A. Marchi of al.

Table 1. fruit growth parameters of field grown Abate Fete1pears at different sampling dates Height (cm)

Samplingdate

1

30 July 1990 20 August 1990 5 September 1990

2 3

Diameter (cm)

10.6a* 12.8b 12.5b

(0.9)’ (1.2) (1.0)

5.58a 6.56b 6.80b

*Means within a row followed by the same letter are not significantly different by protected ‘Standard error

Weight (g) 149.2a 245.8b 247.Ob

I:::{ (0.58)

(21.9) (40.9) (55.1)

Fisher’s least significant difference test @ = 0.05)

Table 2. Amount of CS2 (mg kg-‘) residue on Abate Fete1 fruits measured at each sampling date before and after washing in a

Results

6% sodium silicate solution

Table I shows values for fruit development parameters at each sampling date. The data shows a statistically significant increase in weight (97 g), size (1 cm) and height (2.2 cm) of the fruits between the first and second date. On the contrary, the difference in these parameters between the second and third date was found to be minimal, thus indicating that at the intermediate date the fruit had already reached its maximum development. Residue analysis performed on fruits before and after washing in sodium silicate showed that treatment removed at each date a significant part of the initial dithiocarbamate residue (Table 2). Removal accounted for 67.3%) 77.8% and 58.8% of the initial residue, respectively, for the first, second and third sampling date. In general, after treatment the residue dropped to values below 0.5 CSZ mg kg-‘. For thiram treatments, increasing doses of active ingredient were found to increase CSz residues proportionally, the trend being the same for all three sampling dates (Figure I), with the following linear regression: R2 = 0.988 (p < 0.01) (first date), R2 = 0.974 (p < 0.01) (second date), R2 = 0.980 Cp < 0.01) (third date). Vice versa, at equal doses of treatment, the amount of residue was found to diminish depending on sampling date. For example, treatment with 3.2 g 1-l of active ingredient led to an amount of residue equal to 3.631, 3.329 and 2.887 mg kg-’ at the first, second and third dates, respectively (L.S.D. = 0.292, p = 0.05). The ANOVA showed that both fungicide dose and sampling date significantly affect the variations in the amount of residue detected (Table 3). No significant interaction, however, was found to exist between these two factors. The residues detected on fruits treated with varying doses of metiram (Figure 2) also exhibited the same linear trend: R2 = 0.846 (p < 0.01) (first date), R2 = 0.982 (p < 0.01) (third date). Likewise, at equai doses of active ingredient, the amount of residue decreased from the first to the third date. For example, in fruits treated with 1.6 g 1-l of metiram, the residue was found to be equal to 1.394 mg kg-’ upon the first date and 0.979 mg kg-’ upon the second (L.S.D. = 0.388, p = 0.05). A significant role of dose and sampling date in determining variations in the amount of residue detected was found with ANOVA (Table 3). A comparison of the amount of residue left after treatment with the two fungicides shows, in general, that residue values following treatment with thiram are greater than those following treatment with metiram at equal doses. In the pathogen inoculation tests, an inverse relationship between disease severity and residue levels was

Sodium silicate washing Before After Difference

1

Sampling 2

date

1.50 0.49

1.89 0.42

0.34 0.14

1.01**

1.47**

0.20*

3

Data are average of four replicates *significant at p < 0.01 according to **significant at p < 0.05 according to t test

t test;

"1 3.5 -

32.5 E g N t3

2-

1.5 I0.5 0 j

d I

0

I

I

0.1

0.2

I

0.3

I

0.4

a.i. (%) Figure 1. CS, residue left upon treatment with increasing doses of thiram on Abate Fete1 pears harvested at three growth stages. Vertical bar represents least significant difference (P = 0.05). A = first date, 0 = second date, q = third date.

and the effect of dithiocarbamate dosage and of fruit growth stage on the amount of residue, was determined using the generalised least squares ANOVA procedure.

Means within fungicide treatments were then compared by protected Fisher’s L.S.D. (p = 0.05) whereas the influence of increasing residue values on disease intensity was evaluated by regression analysis. Positive growth responses of S. vesicarium recorded in the plating of fruit washing dilutions were entered in a suitable table (Harris and Sommers, 1968) that gave the corresponding number of microorganisms. The percentage of inhibition of germination from the fungitoxicity test was plotted as probit against the Log concentration of the test compound. Data were analysed using a probit-log concentration progam that gave both the ECsO value and the slope of the response curve, with 95% confidence limits.

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Table 3. Summary of ANOVA for CSp residues on Abate F&t&l fruit treated with either thiram or metiram at varying concentrations of active ingredient at different harvest times Metiram

Thiram

Dose Harvest Dose*harvest Error

df”

Mean square

4 2 8 43

14.885 0.888 0.063 0.042

F value

P>F

df”

Mean square

353.179 21.111 1so2

0.000 0.000 0.185

4 1 4 28

2.866 2.024 0.056 0.061

F value

P>F

46.902 33.115 0.908

0.000 0.000 0.473

“Degrees of freedom

(kO.02 confidence interval, p = 0.05, y = -0.83 + 5.09x) mg kg-’ for metiram. Dilution series of microorganisms from the fruit surface performed immediately after inoculation

43.5 32.5 E 8PI 8

21.5l-

L.S.D. (0.05)0.388

0.5 o-

d I

I

0

0.1

-:. -i.. . 1

1

I

I

0.2

0.3

0.4

a.i. (%)

6

+

Figure 2. CSp residue left upon treatment with increasing doses of metiram on Abate F&I pears harvested at three growth stages. Vertical bar represents least significant difference (P = 0.05) A = first date, q third date.

:

.

0 0

I

I

I

2

I

1

.?

4

CS2 ppm Figure 3. Exponential

found when results were pooled from the different sampling dates (Figures 3 and 4). In particular, with thiram (Figure 3) a marked increase in the residue was seen to be accompanied by a sharp decrease in disease severity, with residue values of about 3-3.5 mg kg-’ of CS2, resulting in a severity index of less than 0.5. The relationship between disease severity and amount with y = of residue ap ears to be exponential, 3 .369-“.69= (y R = 0.797). At the doses of active ingredient employed for treatments, metiram was not found to effectively control the disease (Figure 4). In particular, an increase in the amount of residue from 0.521 mg kg-’ to 2.096 mg kg-’ brought about only a slight reduction of the severity index, which remained at a value of around 2. In this case, the amount of residue and the disease severity class were found to have a low R2 value (R2 = 0.311). Throughout the inoculation tests, control fruit scored disease severity that averaged 2.8 (f0.35). The difference in efficacy of the two dithiocarbamates was also confirmed by in vitro laboratory tests carried out on conidia germinability in the presence of varying concentrations of active ingredient. ECsO values was found to be 2.3 (kO.04 confidence interval, p = 0.05, Y = 4.1 + 2.44.x) mg kg-’ for thiram and 14

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regression of disease severity classes of Stemphylium vesicarium on CS, residues after treatment of Abate F&J pears with vatying concentrations of thiram y = 3.369-“~6*g2X(~ = 0.797).

O i

I

I

I

0

1

2

3

CS2 ppm Figure

4. Linear

Stemphylium

regression

of

disease

severity

classes

of of metiram.

vesicarium on CS? residues after treatment

Abate F&&l pears with varying y = 2.526 - 0.327x (R’ = 0.311).

concentrations

of

Dithiocarbamate

revealed that each fruit hosted on average 500 (+45) propagules of S. vesicarium.

Discussion

Our findings show that the amount of residue detected on Abate Fete1 pear fruits treated with dithiocarbamates depends on: (i) type of product, the amount of residue from thiram treatment tending to be greater than that from metiram at equal doses of active ingredient and under the same experimental conditions; and (ii) treatment time, the amount of residue decreasing as sampling date approached harvest, at equal doses of active ingredient. This latter pattern may reasonably be attributed to some physical change in the skin of the fruit and not to a change in the surface:volume ratio since there was no significant fruit size increase between the second and third sampling dates.

residue and S. vesicarium

infection:

A. Marchi et al.

carbamate-based treatments in the field when climatic conditions are favourabie to infection. Moreover, sprayings in the field result in a non-uniform distribution of the product on the fruit surface and it is likely that some areas on the fruit remain less protected. Another factor that should be taken into account is the amount of residue washed away by rain. In order to work out a more rational treatment schedule, residue degradation times and climatic conditions favouring degradation in the field still need to be determined.

References Blancard,

D., Piquemal,

J. P. and Gindrat, D. (1984) La stemphyliose Hort. 248, ,27-30

de I’asperge. P. H. M.-Rev.

D., Allard, E. and Brest, P. (1989) La stemphyliose pokier ou ‘macules brunes’. Phytoma 406, 37-38

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A., Rovesti, L., Di Marco, S. and Ponti I. (1986) Attivita di bruna del pero. Rivistu di Frutticoltura 1, 51-54

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J. P. and Humbert, W. (1991) Stemphyliose du poirier: Etude de la biologie du parasite et recherches des fongicides actifs. Cugier,

Percentage removal of dithiocarbamate field residues was similar to that previously reported by Folchi et al. (1993) of around two-thirds of the initial residue. This residue left on the fruit did not affect disease development since inoculated fruit that did not receive any post-harvest treatment always scored high values of disease severity . As far as the possibility of preventing infection by S. vesicarium is concerned, thiram was found to be much more effective than metiram, these findings are similar to those obtained from fungitoxicity tests and in field trials (Brunelli et al., 1986). Crop protection afforded by thiram, even if high, was not total. In fact, in some cases, despite the high doses of product employed, disease was detected, even if only at low level. In this regard, however, account should be taken of the fact that the experimental conditions adopted during fruit incubation, as well as the high inoculum density employed, were particularly favourable to disease development. As a matter of fact, dilution series of microorganisms from the fruit surface performed immediately after inoculation revealed that each fruit hosted a high number of pathogen propagules. Under the conditions employed for our experiments, the dose of thiram recommended for field treatments (0.7-0.8 gl-’ active ingredient), corresponding to a residue of less than 1 mg kg-‘, does not guarantee effective fruit protection. The efficacy of this treatment in the field presumably depends on residue accumulation resulting from repeated treatments. Nonetheless Ponti et al. (1993) showed that in years where high pathogen inoculum densities are present in the orchard or when climatic conditions are particularly conducive to the disease, even stategies based on frequent treatment schedules may result in unsatisfactory control of the disease. Our data showed that with favourable infection conditions, such as those of the in vitro tests, the fruit may be expected to be subject to pathogen infection below the CS2 threshold level of 3-3.5 mg kg-‘. This high capability of the pathogen to infect the fruit even in presence of relatively high CS2 residue could explain the low efficacy of dithio-

Phytoma 431, 47-50 P. G., Falloon, L. M. and Grogan, R. G. (1987) Etiology and epidemiology of Stemphylium leaf spot and purple spot of asparagus in California. Phytopathology 77, 407413

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C., Shucker& R. and Bornak, W. (1967) Improved method for the determination of Ethylene-bis-dithiocarbamate residues in plants, fruits and vegetables. J. A. O.A. C. 40, 1102-l 108

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R. F. and Sommers, L. E. (1968) Plate-dilution frequency technique for assay of microbial ecology. Appl. Microbial. 16, 330Harris, 334 Hocking, A. D. and Andrews, S. (1987) Dichloran chloramphenicol peptone agar as an identification medium for Fusarium species and some dematiaceous Hyphomycetes. Trans. Br. Mycol. Sot. 89, 239-

246 G. E. (1971) Collaborative study of the determination of dithiocarbamate residues by a modified carbon disulfide evolution method. .I. A.O.A.C. 54, 528-532 Keppel,

Martini, A., Federici, F. and Rosini, G. (1980) A new approach to the study of yeast ecology of natural substrates. Can. J. Microbial. 26, 856-859 Miller, M. E., Taber,

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J. A. (1978) Stemphylium

blight of onion in south Texas. Plant Dis. 62, 851-853 E. and Vilardell, P. (1992) Evaluation of FAST as system for scheduling fungicides sprays for control of Stemphylium vesicarium on pear. Plant Dis. 76, 1221-1226 Montesinos,

forecasting

Ponti, I., Cavanni, P. and Brunelli, A. (1982) ‘Maculatura bruna’ delle pere: eziologia e difesa. Informatore Fitopatologico 3, 35-40 Ponti, I. and Cavanni, P. (1983) lndagine preliminare sulla fitotossicita colturali di Stemphylium vesicarium, agente della ‘maculatura bruna’ del pero. Informatore Fitopatologico 9, 55-57 di filtrati

Ponti, I., Brunelli, A., Tosi, C., Basaglia, M., Bevilacqua, T., Emiliani, G., Cont, C. and Viccinelli, R. (1993) Verifica dell’attivita di diversi preparati contra la maculatura bruna del pero. Informatore Fitopatologico 5, 45-52 Porta-Puglia,

A. (1981) Stemphylium

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per la Patologia Vegetale

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Vilardell, P. (1988) Stemphylium vesicarium en plantaciones de peral. Fructicultura Profesional 18, 51-55

Received

1 March 1994 Revised 12 September 1994 Accepted 12 September 1994