IE62521B1 - Use of 2, 2-dibromo-3-nitrilopropionamide as a crop sporicide or fungicide e.g. by fumigation - Google Patents

Abstract

Fumigant compositions comprising 2,2-dibromo-3-nitrilopropionamide (DBNPA) which are useful in treating crops against fungal and bacterial infections are disclosed. Sporicidal and fungicidal compositions comprising 2,2,-dibromo-3-nitrilopropionamide (DBNPA) and the use of DBNPA against pathogenic microorganisms which attack potatoes are also described.

Description

USE OF 2, 2-DIBROMO-3-NITRILOPROPIONAMIDE AS A CROP SPORICIDE OR FUNGICIDE E.G. BY FUMIGATION The present invention relates to a fumigator for 5 treating crops against fungal and bacterial infections «-< by fumigation, to a method of treatment of crops by fumigation and to the use of 2,2-dibromo-3-nitrilopropionamide (DBNPA) as a sporicide or fungicide in the treatment of crops. 1 0 One of the crops most suffering from both fungal and bacterial attack is potatoes, to which particular attention has been given in the art, and which will be discussed in detail in the following description.

Potato diseases are caused by fungi and bacteria. These microorganisms attack both grown and seed potatoes, and throughout this specification, whenever reference is made to potatoes, it is understood that both grown and seed potatoes are meant, as applicable. Numerous factors influence the amount of disease expression in a potato crop, such as environmental conditions, interaction between different pathogenes, cultivar resistance or succeptibility, plant nutrition, seed handling, etc. Many efforts have been directed to the control of potato diseases, because of the economic value of this crop; A large number of biocidals is employed to treat potato crops, notably the organo-mercury compounds. These, as well as other commonly , employed biocides, present severe drawbacks, since they are toxic and ·» phytotoxic. Among the best known fungicidal compounds, Caspan (3-methoxyethylmercury chloride) and Captan (tetrahydro2-[(trichloromethyl)thio]-4-cyclohexene-l,2 -dicarboximide) are commonly employed in the art.

Among the most common microorganisms which attack potatoes, 5 there are found Erwinia carotovora, Streptomyces scabies, Fusarium oxysporum, Verticillium dahliae and Rhizoctonia solani. Among these, Erwinia car. - which is a major responsible for potato diseases and Streptomyces scabies are bacteria, and Fusarium oxysporum, Verticillium dahliae and Rhizoctonia solani are fungi.

Few of the chemicals used on potatoes have appreciable volatility and fungicidal activity is often confined to sites of initial deposition, so that total cover of the tuber is essential. This is of course a severe drawback which reflects on the cost and quality of potato treatments. Several methods of application are known in the art. Total immersion of tubers is often employed which, however, is hazardous because of the risk of soft rot in the treated tubers and blackleg in the crop. Dusting by shaking or rolling in fungicide powder is often employed. This method, however, is relatively complex and the amount of fungicide reaching the tuber is dependent on the inert carrier. Spraying of mists is also employed. This method is technically complex, requires rotation of tubers, the spray liquid is often a suspension and relatively large amounts of spray are required. ο ο Among the chemicals normally employed for the control of fungal and bacterial diseases the organo-mercury compounds are often employed to treat potatoes. These compounds have the considerable drawback of being toxic and phytotoxic and, according to recent works (Logan, C.: Annual Report on Research and Technical Work of the Department of Agriculture for Northern Ireland, 198-200 (1984)) they do not provide satisfactory protection against soil-borne black-leg pathogenes.

Fumigation is a method which overcomes many of these drawbacks, besides being simple and economic. The problems connected with fumigation are the low volatility of the commonly employed chemicals and that they are harmful in vapour form. The art has therefore long felt a want of a fumigant formulation which is relatively non-hazardous and which can be employed in crop fumigation.

DBNPA has been known for some time to possess bacteriocidal activity. U.S. Patent No. 2,419,888 discloses the bacteriocidal and fungicidal activity of a class of compounds which includes DBNPA. DBNPA, however, is not specifically disclosed or exemplified in this patent. U.S. Patents Nos. 4,285,765 and 4,604,405 disclose synergistic antimicrobial mixtures of DBNPA and other active materials. According to recent studies (G. Krizmann et al., Gan, Sade Vemeshek, Israel, July 1985) DBNPA was found to possess no fungicidal activity against fungi which attack potato crops. The above authors carried out extensive in vitro and field experiments with fungi which attack potato crops, and on the basis of their results concluded that DBNPA does not possess any fungicidal activity against such fungi.

It has now been surprisingly found that DBNPA, notwithstanding its exceedingly low volatility, can also be conveniently employed as a fumigant, and that fumigation therewith provides an excellent biocidal effect, both against fungi and bacteria. It has further been found that fumigation with DBNPA is particularly effective against fungi and bacteria which are responsible for potato diseases. Another surprising discovery is that it is possible to provide formulations containing DBNPA whose efficacy is not limited to normal biocidal and fungicidal activity and to fungistatic activity, but which also exhibit sporistatic and sporicidal activity.

By fumigation is meant the delivery of biocidally effective amounts of DBNPA to the locus to be treated against microorganisms from a reservoir containing DBNPA, either alone or in admixture with solvents and/or other biocidal or biocidally inert compounds, through a gas as the transfer medium, without any direct contact between the material contained in the reservoir and the locus to be treated.

Comparatively few chemicals are sporicidal, and many powerful bactericidal agents, such as phenols and quaternary ammonium compounds, have little effect on the viability of bacterial spores [see, e.g., S. S. Block, Disinfection, Sterilization and Preservation, Leo & Febiger, 1983, p. 739J.

By sporostatic activity it is meant that germination of spores is inhibited, as long as an effective concentration of an active material is present, but germination takes place after no active material is present any more. A sporicidal is defined as an agent that destroys microbial spores. - 6 In one aspect, the invention provides a fumigator comprising a reservoir in which there is present a biocide which is 2,2-dibromo-3-nitrilopropionamide or a composition containing 2,2-dibromo-3-nitrilopropionamide in admixture with an organic solvent, water or a mixture of water with an organic solvent, the arrangement being such that in use the biocide is delivered from the reservoir to the locus to be treated through gas as the transfer medium without any direct contact between the material present in the reservoir and the locus to be treated .

In a further aspect the invention provides a method of treating crops infected or liable to be infected with microorganisms to . inhibit or prevent growth of said microorganisms and/or germination of their spores, characterised in that the crop to be treated is fumigated with a biocide which ’is 2,2-dibromo-3-nitrilopropionamide or a composition containing 2,2-dibromo-3-nitrilopropionamide in admixture with an organic solvent, water or a mixture of water with an organic solvent.

In a third aspect the invention relates treating crops infected or liable to be infected with microorganisms to inhibit or prevent growth of said microorganisms 6a and/or germination of their spores, characterised in that the crop to be treated is fumigated with a biocide having the features set forth above.

The organic solvent must of course be usable with the crops involved. For instance, if edible crops are treated, the solvent must be such that it does not remain for long periods on the crop and is not toxyc or phytotoxic. Preferably, the organic solvent is a glycol selected from mono-, di- or poly-propylene glycol, more preferably, dipropylene glycol.

A preferred fumigant composition of the invention comprises: 0 to 100 wt% of 2,2-dibromo-nitrilopropionamide, preferably about 20 wt%; to 80 wt% organic solvent, preferably about 60%; and to 80 wt% water, preferably about 20 wt%.

A crop particularly suitable for being treated by the method of the invention is potatoes or seed potatoes.

Compositions not comprising an organic solvent, while retaining their sporicidal properties, are found to be generally less effective than compositions which do comprise an organic solvent.

( As will be apparent to a person skilled in the art, an organic solvent, . f . in order to be suitably employed to carry out the invention, must be such that DBNPA is at least partially soluble therein, and at the same time must not be phytotoxic - or toxic, since the crops involved are edible.

Preferably the organic solvent is a glycol and more preferably the glycol is selected from mono-, di- or polypropylene glycol, more preferably di-propylene glycol (DPG).

According to a preferred feature of the invention, a bioriHa.L.composition contains: a) 5 to 25 wt% 2,2,-dibromo-3-nitrilopropionamide; b) 0 to 95 wt% of water; and c) 0 to 95 wt% of a glycol.

Most preferred compositions for use in the invention contain: a) about 20 wt% 2,2,-dibromo-3-nitrilopropionamide; b) about 20 wt% of water; and c) about 60 wt% of 311 organic solvent e.g. di-propy lene glycol.

The method for treating potatoes and/or seed potatoes against fungal and bacterial potato diseases according to the invention is characterized in that a composition as defined above is applied to the potatoes or seed potatoes, whenever fungi and/or bacteria or their spores are to be destroyed, or their growth or germination inhibited. Application of the composition can be effected in any suitable way, but spraying and fumigation are most preferred.

It is readily understood that the surprising efficacy of the compositions used in the invention against fungi which attack potato crops, together with the known activity of the active material against a variety of bacteria - and its sporicidal activity, form the basis for a very convenient use thereof in the treatment of crop diseases, and particularly potato diseases which arise from a combination of different microorganisms.

The microorganism may be any microorganism which attacks crops, and particularly the method can be conveniently carried out to destroy Erwinia carotovora or Streptomyces scabies or Fusarium oxysporum or Verticillium dahliae or Rhizoctonia solani, or two or more of the said microorganisms present on a given crop at the same time.

The above and other characteristics and advantages of the invention will be better understood through the following illustrative and non limitative examples. In all cases the high temperature (>37°C) experiments were carried out in a fumigation cell having a total volume of 15 lit., containing a controlled heating unit located at the centre of an insulating block. Two aluminium plates (3 ml volume each) were placed on the heating unit. The agar plates were placed above the heating unit at room temperature. The biocidal formulation was heated to the chosen temperature, and the plates were then placed in the closed cell for the required period. After removing the plates, they were incubated at 27°C for 24 hours, and viable counts were compared to those of parallel control experiments. Experiments at lower temperatures were carried out in a fumigation .vessel maintained throughout at the designated temperature, as will be further described in the examples. Compositions containing a glycol also contained 0.1% buthylated hydroxytoluene (BHT) as an antioxidant.

Example 1 Fumigation experiments were carried out in the fumigation cell, to test the efficacy of two different formulations of DBNPA against Erwinia carotovora ATCC 15713, at different temperatures. The results of these tests are shown in Table I. The fumigant compositions employed were a solid form, comprising DBNPA alone, and a liquid formulation containing dipropylene glycol (DPG) (20:20:60 wt% DBNPA:WATER:DPG), marked (s) and (1) in Table I respectively. - 20 Liquid formulations containing a glycol also contained in each case 0.1% buthylated hydroxytoluene (BHT) as antioxidant. As can be appreciated from Table I, the efficacy of the liquid formulation is appreciably higher than that of the solid form, and a sensible effect of ( the fumigation cell temperature can be seen. The total weight of the active material is not important, as long as some active material remains at the end of the experiment, and the important factor is its evaporation rate. The actual weight of the active material in liquid formulations can be calculated on the basis of the amounts employed in each case.

I Example 2 Example 1 was repeated at low temperatures (4, 27 and 37°C), and the results of these tests are shown in Table II. The Erwinia spores were placed in shake flasks containing 50 ml cell suspension, and vials containing the biocide formulation were hanged above the water level, within the fumigation vessel. As it is seen from the comparison of Tables I and II, longer contact times are required for lower fumigation temperatures.

Examplg-3.

The efficacy of DBNPA against spores of three fungi was tested in a fumigation cell. The fungi employed were Fusarium oxy., Verticillium dahliae and Rhisoctonia sol.. Also spores of one bacteria (Streptomyces scabies) was tested for comparison. The results are reported in Table III. The experiments were carried out in a ( fumigation cell having a total volume of 15 lit., containing a controlled heating body located at the centre of an insulating block. Two aluminium plates (3 ml volume each) were placed on the heating body. The agar plates were placed above the heating unit at room temperature. The biocidal formulation was heated to the chosen temperature, and the plates were then placed in the closed cell for the required period. After removing the plates, they were incubated at 27°C for 24 hours, and viable counts were compared to those of parallel control experiments.

All experiments were carried out at 70°C and with different initial inoculum. The fumigation cell was charged with a 20:20:60 wt% DBNPA:WATER:DPG formulation. In all cases no survivors were found, for all fungi tested.

Example 4 A single sample of a 20:20:60 formulation of DBNPA in DPG and water was used for a long-term fumigation experiment, in order to test the long term activity of such a formulation. The fumigation experiment was carried out at 70°C, with contact times of 1 hour for each plate, the plates being inoculated with Erwinia carotovora. Two identical samples of the formulation were employed in the experiment. The results of this test are shown in Table IV. The cell ( was operated for an overall 61 hours and retained its activity against Erwinia during 55 hours. Water was seen to evaporate quickly and after the first 5 hours a make-up of water was added to the formulation. The weight loss per hour of an hydrated sample was about 20%/hour, while the average weight loss of a dehydrated sample was 1.5%, which accounts for an evaporation rate of DBNPA of less than 0.4%/hour (about 30 mg/hr), if the evaporation rate is taken as constant.

Example 5 A number of experiments were carried out, in order to test the relative efficacy of different mixtures of solid DBNPA and water. The results of these tests are shown in Table V. The weight losses are reported for each of the two . identical samples of aqeous formulation employed together in the experiment.

The fumigation temperature was kept at 70°C throughout the experiments. The best results were obtained with compositions containing 20 wt% DBNPA in water. This formulation provided results sensibly better than any solid/aqueous formulation tested, and similar to the results of the formulation containing DPG. It should also be noted that the evaporation rates in two identicall cells differ within unpredictable limits. This is believed to be due to the non-homogeinity of a formulation of DBNPA in water alone.

Example.g Fumigation experiments were carried out with several ingredients of the DBNPA formulations and with DBNPA analogues, in order to measure their fumigation biocidal activity and compare it with the fumigation efficacy of the compositions of the invention. All fumigation experiments were carried out at ,70°C, and two microorganisms were tested: Erwinia carotovora subsp. carotovora (ATCC 15713), and spores of Fusarium oxysporum (isolated from contaminated crops).

The results are summarized in Table VI. The fumigant compositions tested, as identified in the table, were as follows: DPG - pure dipropylene glycol; EG - pure ethylene glycol; ®r2 a formulation of 7% Br2 and 23% H2O in 70% DPG; Monobromo - a formulation of 25% monobromocyanoacetamide (MBNPA,- prepared by mixing equimolar quantities of DBNPA and cyanoacetamide), 19% H2O and 56% DPG; DBNPAZEG - a formulation containing 20% DBNPA, and 20% H2O2 in 60% EG; DBNPA/DPG - a formulation containing 20% DBNPA, 20% H2O and 60% DPG; The percentages given above are by weight.

The two solvents DPG and EG did not show any biocidal activity under 5 the test conditions. The Br£ formulation exhibited some biocidal properties, giving a partial control with long contact times (1 hour).

The DBNPA/EG formulation controlled Erwinia car. to some extent, when a contact time of 1 hour was examined, and controlled low inoculum of Fusarium oxy. (8x10® spores/plate) with a contact time of half an hour, and the high inoculum (8x107 spores/plate) using a contact time of 1 hour. DBNPA/DPG was effective against both Erwinia sp. and Fusarium oxy., after 15 minutes of fumigation.

Example 7 Activity of DBNPA Against Streptomyces scabies and Fusarium oxv.

The efficacy of DBNPA was tested against Streptomyces scabies and Fusarium oxy., as compared with the efficacy of the known biocidal Captan. In vitro experiments were carried out to determine the relative efficacy of DBNPA (20% formulation in 60% DPG and in water) and Captan. In all experiments a basically unlimited contact . I time was employed, and the ability of the various spores to germinate in the presence of the active ingredient was examined. Spores that did not germinate within 10 days were considered as destroyed. The data reported under cfu/μΙ refer to the germination of spores taken from the original flask after 7 to 10 days, and placed under germination conditions on agar plates. The nutrient solution in the tests was malt for Streptomyces scabies and Czapek-Dox for all other microorganisms employed.

The results of these experiments are summarized in Tables VII and VIII. It can be noted that 200 ppm of DBNPA were required to inhibit Ιθ spore germination of Streptomyces scabies, at the 10^θ inoculum, under which conditions 5 spores/10 μΐ germinated on agar plates. 500 ppm were biocidal. No substantial difference was detectable between the activities at 27°C and 4°C. 100 ppm were biocidal for the 10θ inoculum of Fusarium oxy..

In comparison, 500 ppm of Captan were biostatic under the same conditions, but the spores still germinated on agar plates. With Fusarium, 500 ppm were inhibiting both at 27°C and 4°C.

Example 8 Activity of DBNPA at Low Concentrations Against Fusarium oxy, sp. and Verticillium dahliae sp.

( ; The efficacy of DBNPA was tested at low concentrations (25-100 ppm) against Fusarium oxy. sp. and Verticillium dahliae sp., as compared with the efficacy of the known biocidal Caspan. In vitro experiments were carried out to determine the relative efficacy of DBNPA (20% formulation in 60% DPG and in water) and Caspar In all experiments a basically unlimited contact time was employed, and the ability of the various spores to germinate in the presence of the active ingredient was examined, as hereinbefore detailed.

Three levels of initial inoculum were examined: 25 ppm of DBNPA formulation were sufficient to inhibit germination at the low inoculum, and 50 ppm at the high inoculum. In comparison, even 100 ppm of Caspan did not inhibit the germination of the low initial inoculum of Fusarium, but did inhibit the germination of the low inoculum of Verticillium. 100 ppm was not enough to prevent germination of the high inoculum of spores of any of these fungi. The results of these experiments are reported in Tables IX and X.

Example 9 Determination of the Minimal Inhibitory Concentration (MIC). Experiments were carried out to determine the minimal inhibitory concentration of DBNPA, as compared with Captan and Caspan, for three fungi: Trichoderma sp. (which is a beneficial soil fungus that does not attack potatoes), Fusarium oxy. sp. and Verticillium dahliae sp. The MIC values for these fungi and active materials are reported in Table XI.

These data illustrate an important advantage of the invention. Comparison of the data shows that the MIC of DBNPA for Trichoderma sp. is very high (200 - 500) as compared to the MIC for Fusarium oxy. sp. (25) or Verticillium dahl. sp. (25 - 50). This means that these last two harmful microorganisms can be effectively treated, without attacking Trichoderma sp.. Trichoderma is a beneficial fungus, because it attacks other fungi which are damaging to crops.

Therefore, its presence in the soil is very desirable. In contrast, the corresponding MICs of, e.g., Captan, are of the same order of magnitude: 500 - 1000 for Trichoderma, and 200 - 500 for Fusarium. Therefore, a successful treatment of Fusarium employing Captan will result in the distruction of the beneficial fungus Trichoderma.

Such an occurrence is mostly avoided when operating according to the invention.

Example 1-Q, The sporicidal activity of DBNPA formulations was compared with that of Captan and Caspan, against spores of Streptomyces scabies sp. • 20 (isolated from injured crops). The experiments were carried out in liquid cultures and the results analyzed as in the previous examples.

The formulations of DBNPA employed were: A) a 20:20:60 wt% formulation of DBNPA:WATER:DPG; and B) pure solid DBNPA dissolved in water. The original inoculum was in all cases 10? spores/ml. The results of these experiments are detailed in Table XII.

The results of Table XII clearly show the superior sporicidal activity of DBNPA which, in both formulations, was found to exhibit sporicidal or even sporistatic activities at concentrations of 100 ppm, while no sporicidal activity was shown by Caspan and Captan even at 300 ppm.

Example 11 Example 10 was repeated, using Fusarium oxy. so. as the tested microorganism. In this test the relative efficacy of the three biocides employed was tested on mycellia as well, and concentrations of glucose left in the medium were measured after 24 hours, while the dry weights were measured after 48 hrs. The concentration of glucose left in the medium is a measure of the growth that took place, since glucose is consumed by Fusarium oxy. sp. in the medium. Thus, the control sample, to which no biocide was added, was seen to consume all the glucose during growth. The results are summarized in Table XIII for spores, and in Table XIV for mycellia. The No. of Survivors for spores which germinated into mycellia was of course not measurable, and is designated in the table as not measured.

It can be seen from the results in Table XIII that both formulations of DBNPA tested exhibited high sporicidal and sporistatic activity. Using an inoculum of 1 χ 10θ spores/ml, 30 ppm of DBNPA formulation A were sporistatic, and 45 ppm were sporicidal, while 60 ppm of either Caspan or Captan were not even sporistatic. This is surprising since, as shown in Table XIV, Captan exhibited a fungicidal activity superior to that of DBNPA, against mycellia of Fusarium oxy. sp., and exhibited at 200 ppm an activity similar to that of 400 ppm of DBNPA. Caspan was the least effective fungicidal among the three tested.

It is clear from the above description that formulations according to the invention containing DBNPA provide a very effective means to destroy spores and fungi, and in particular those fungi which attack potato crops and their spores. While the methods and compositions herein described have been found to be convenient in practical use, many variations can be effected, as will be apparent to the man of the art. For instance, different solvents, proportions and additives can be employed, and different crops can be treated or different fungi destroyed, or different fumigation cells and methods can be employed, and different microorganisms destroyed, or different solid or liquid formulations used, all without exceeding the scope of the invention.

Tabled Efficacy of DBNPA Formulations against Erwinia in Fumigation Initial count Temp. .cq Contact time (b) Biocide content No. of survivors per plate 5Ί07 50 4 23.20 gr (s) 5-107 5Ί05 50 4 23.20 gr (s) 5-105 5Ί03 50 4 23.20 gr (s) 5Ί03 6Ί07 100 4 23.20 gr (s) no survivors 6Ί05 100 4 23.20 gr (s) no survivors 6Ί03 100 4 23.20 gr (s) no survivors 1Ί07 70 2 23.20 gr (s) ~ 107 1Ί05 70 2 23.20 gr (s) .103 1Ί03 70 2 23.20 gr (s) 10 8Ί06 70 4 6 ml (I) no survivors 8104 70 4 6 ml (I) no survivors 8Ί02 70 4 6 ml (I) no survivors 4Ί07 70 2 6 ml (I) no survivors 4Ί05 70 2 6 ml (I) no survivors 4Ί03 70 2 6 ml (I) no survivors 3Ί08 70 1 6 ml (I) no survivors 3Ί06 70 1 6 ml (I) no survivors 3-104 70 1 6 ml (I) no survivors 2Ί08 70 0.5 6 ml (I) .10® 2-106 70 . 0.5 6 ml (I) -10® 2-104 70 0.5 6 ml (I) -103 4-107 90 1 23 gr(s) -10® Table I (Continued) Initial count Temp. rQ Contact time (h) Biocide content No. of survovors per plate 4Ί05 90 1 23gr(s) .10* 4-103 90 1 23 gr (s) ~T02 4-107 90 1 6 ml (I) no survivors 4-105 90 1 6 ml (I) no survivors 4-103 90 1 6 ml (I) no survivors 4-107 90 0.5 6 ml (I) no survivors 4-105 90 0.5 6 ml (I) no survivors 4-103 90 0.5 6 ml (I) no survivors IabiaJl Fumigation of Erwinia at Room Temperature initial count Temp JCQ Contact time..(hrs) Biocide No. of Survivors per plate 2x108/c’c 37 24 1.5 gr (s) 3x108 2x107/c'c 37 24 1.5 gr (s) 3x108 2x108/c'c 37 24 3.0 gr(s) 1x108 2x107/c’c 37 24 3.0 gr (s) 5x107 5x107/c’c 37 24 3.0 ml (I) no survivors 5x107/c'c 37 24 4.5 ml (I) no survivors 5x107/c’c 37 24 6.0 ml (I) no survivors 9x106/c'c 27 24 3.0 ml (I) no survivors 9x106/c'c 27 24 4.5 ml (I) no survivors 9x106/c’c 27 24 6.0 ml (I) no survivors 4x107/c’c 4 96 3.0 ml (I) no survivors 4x107/c’c A 96 4.5 ml (I) no survivors 4x107/c'c 4 .96 6.0 ml (I) no survivors Iabkdii Efficacy of a DBNPA Formulation Against Various MQ^ Type of MQ Fusarium oxy. Temp m 70 70 70 70 Contact time fh) 1 0.5 0.5 1 Inoculum efu /plate 3x105 3x 105 1 x107 1 x107 Survivors cfu/plate Streptomyces scabies 70 1 2x105 2x103 70 1 2x104 2x 102 70 1 2x103 2x 10 70 0.5 2x105 2x105 70 0.5 2x104 8x103 70 0.5 2x103 2x103 Verticillium dahliae 70 0.5 5x105 - 70 0.5 5x103 - 70 0.5 5x10 - 70 1 5x105 . 70 1 5x103 - 70 1 5x10 - 70 0.25 6x105 - 70 0.5 6x 105 - Table III (Continued) Type of MO Temp m Contact limalhl Inoculum cfu/plate Rhizoctonia sol. 70 1 mycellia 0.5 cm covered plate Survivors cfu/plate lafale.JV.

Fumigation of Erwinia Carotovora with a Single Sample of Fumigant Overall fumigation time (hrs) Initial weight (gr) Average weight loss per hour (%) Initial count cfu /Dlate Survivors 1 · Δ 3.44 R 3.44 19 1x108 2 2.83 2.78 1.2 1 x108 - 3 2.76 2.77 0 1 χ 108 - 4 2.79 2.73 1.2 1 x108 - 5 2.73 2.72 0.5 1 x108 - 6‘ 3.49 3.52 19 1 x108 - 7-12 2.33 1.25 1.5 1 x108 - 13-18 2.14 1.13 1.9 1x108 - 19-24 2.04 1.03 2.3 1 x108 - 25-28 1.81 0.89 1.9 1 x107 - 29-32 1.74 0.82 1.8 1 x107 - 33-35 1.70 0.78 2.3 9x107 - 36-42 1.64 0.73 1.8 5x107 - 43-49 1.50 0.65 2.6 3x107 - 50-55 1.35 0.53 1.33 3x107 - 56-61 1.28 0.50 1 3x107 + * 20% water was added to the residue prior to fumigation -Table V Eumiaation of Erwinia Using Solid DBNPA in Water Formulation Mixture Contact solid time Inoculum Survivors Weight d2O (%) DBNPG(%) (hours) (cfu /Dlate) (cfy-Zpl^tQ) loss (%) 20 80 0.5 5x108 5x108 - 50 50 0.5 5x108 5x108 41,50 50 50 1 4x108 50% kill 26, 18 50 50 1 4x108 80% kill 26,18 50 50 1 4x 108 50% kill 14,31 50 50 1 4x106 no survivors 26,18 80 20 1.5 4x108 no survivors 51,58 80 20 0.5 4x108 no survivors 4,17 80 20 1 4x108 no survivors 57, 80 20 80 1 2x109 2x108 22,19 20 80 1 2x107 no survivors 20 80 0.5 2x109 2x 109 17, 20 20 80 0.5 2x107 2x106 50 50 1 2x109 2x107 49,25 50 50 0.5 2x109 2x108 37, 50 50 50 0.5 2x107 2x106 80 20 0.5 2x109 no survivors 54, 31 80 20 0.5 2x107 no survivors 54, 31 80 20 1 2x109 no survivors 50, 61 Table VI Comparison of the Fumigation Efficacy of Various Ingredients and analogues of DBNPA formulations, at 70°C Active Type Contact time Inoculum spores or cfu Weight loss Number of survivors Inaredient of MO (hours) 0.5 J2£Lp]£l£ 1x104 % cfu/Dlate DPG Erwin. Not detectable Grows like control DPG Erwin. 1 1x104 Not detectable Grows like control DPG Fus. oxy. 0.5 2x106 Not detectable Grows like control DPG Fus. oxy. 1 2x106 Not detectable Grows like control 53 Erwin. 0.5 1x104 Not detectable Grows like control 53 Erwin. 1 1x104 Not detectable Grows like control 53 Fus. oxy. 0.5 2x10® Not detectable Grows like control 53 Fus. oxy. 1 2x10® Not detectable Grows like control Br2 Erwin. 0.25 1x104 9% Growth Br2 Erwin. 0.5 1x104 Not measured Growth Br2 Erwin. 1 1x104 Not measured Growth Br2 Fus. oxy. 0.25 2x10® 9% Grows like control Br2 Fus. oxy. 0.25 2x104 9% Grows less than control Br2 Fus. oxy. 0.5 2x10® Not measured Some growth Br2 Fus. oxy. 0.5 2x104 Not measured Some growth Br2 Fus. oxy. 1 2x10® Not measured Some growth Br2 Fus. oxy. 1 2x104 Not measured Some growth Monobromo Erwin. 0.25 1x104 7% Some growth Monobromo Erwin. 0.5 1x104 10% Some growth Monobromo Erwin. 1 1x104 11% 45 Monobromo Fus. oxy. 0.25 2x10® 7% Some growth Monobromo Fus. oxy. 0.5 2x10® 10% Some growth Monobromo Fus. oxy. 1 2x10® 11% No growth DBNPA/EG Erwin. 0.25 1x108 4% Grows like control DBNPA/EG Erwin. 0.5 1x10® 11% 10 Table VI (Continued) Active Type Contact time Inoculum spores or cfu Weight loss Number of survivors Ingredient DBNP/VEG of MO jttlfiuisl 1 Der Dlate 1x108 % cfu/plate Erwin. 3% Some growth DBNPA/EG Fus. oxy. 0.25 8x107 4% Growth DBNPA/EG Fus. oxy. 0.25 8x105 4% 10 DBNPA/EG Fus. oxy. 0.5 8x107 11% 1 DBNPA/EG Fus. oxy. 0.5 8x105 11% 11 DBNP/VEG Fus. oxy. 1 8x107 3% No growth DBNPA/EG Fus. oxy. 1 8x105 3% No growth DBNPA/DPG Erwin. 0.25 1x10® 2% No growth DBNPA/DPG Erwin. 0.5 1x108 16% No growth DBNPA/DPG Fus. oxy. 0.25 8x107 2% No growth DBNPA/DPG Fus. oxy. 0.5 8x107 16% No growth Table VII Efficacy of a DBNPA Formulation in Liquid Cultures Survivors M2 Temp. m Biocide cone. (ppm), Inoculum Spores/ml Growth in original flask-,. Streptomyces scabies 27 100 1x101° growth Streptomyces scabies 27 200 1x101° no growth Streptomyces scabies 27 500 1x101° no growth Streptomyces scabies 4 100 1x101° growth Streptomyces scabies 4 200 1x1010 no growth Streptomyces scabies 4 500 1x101° no growth Fusarium oxy. 27 100 1x109 no growth Fusarium oxy. 27 200 1x109 no growth Fusarium oxy. 27 500 1x109 no growth Fusarium oxy. 4 100 1x109 no growth Fusarium oxy. 4 200 1x109 no growth Fusarium oxy. 4 500 1x109 no growth IahieYlil Efficacy of Captan in Liquid Cultures Survivors M2 Temp. (°C) Biocide cone. (DDm) Inoculum SDores/ml Growth in original flask cfu/ul Streptomyces scabies 27 100 1x101° growth - Streptomyces scabies 27 200 1x101° no growth 60 Streptomyces scabies 27 500 1x101° no growth 15 Streptomyces scabies 4 100 1x101° growth - Streptomyces scabies 4 .200 1x101° growth - Streptomyces scabies 4 500 1x101° growth 50 Fusarium oxy. 27 100 1x109 . ( growth Fusarium oxy. 27 200 1x109 growth - Fusarium oxy. 27 500 1x109 no growth - Fusarium oxy. 4 100 1x109 growth - Fusarium oxy. 4 200 1x109 growth - Fusarium oxy. 4 500 1x109 no growth - .Table IX Efficacies of a DBNPA formulation and Caspan on Fusarium oxy, sp, Initial Count (Spores/ml) Biocide Biocide gQng, (ppm) Growth 6x 105 DBNPA 25 - 6x105 DBNPA 50 - 6x105 DBNPA 100 - 6x106 DBNPA 25 some growth after 96 hrs 6 χ Ϊ06 DBNPA 50 - 6x106 DBNPA 100 - 1 x108 DBNPA 25 - 1 x108 DBNPA 50 - 6x 105 Caspan 25 + after 24 hrs 6x105 Caspan 50 + after 24 hrs 6x 105 Caspan 100 + after 96 hrs 6x 106 Caspan 25 + after 24 hrs 6x106 Caspan 50 + 6x106 Caspan 100 + 1 x108 Caspan 25 + 1 x108 Caspan 50 + Table X Efficacies of a DBNPA formulation and CasDan on Verticillium dahliae Initial Count Biocide (Spores/ml) Biocide cone, (ppm) Growth 6x10® DBNPA 25 - 6x10® DBNPA 50 - 6x10® DBNPA 100 - 6x10® DBNPA 25 some growth after 96 hrs 6x10® DBNPA 50 - 6x 10® DBNPA 100 - 6x10® Caspan 25 + after 96 hrs 6x10® Caspan 50 + after 96 hrs 6x10® Caspan 100 - 6x10® Caspan 25 + 6x10® Caspan 50 + 6x10® Caspan 100 + (.

Table XI Minimal Inhibitory Concentrations M2 Biocide Inoculum spores/ cfu/μΙ Temp. m MIC (ppm active material) Trichoderma DBNPA 106 27 200 sp. ti n ft 106 4 200 Fusarium oxy. sp. tl 6x106 27 25 Verticillium dahl. sp. n 6x106 27 25 Trichoderma Captan 106 27 500 tl tl tl 106 4 200 Fusarium oxy. sp. tl 1 x109 27 200 n ti tl 1x109 4 200 Fusarium oxy. sp. Caspan 6x106 27 >100 Verticillium dahl. sp. n 6x 106 27 >100 Biocide Formul. A Formiil. B Caspan Caspan Caspan Captan Captan Tah!fi_X!l Efficacy of Formulations A and B, Captan and Caspan on Spores ι of Streptomyces scabies sp. Concentration No. Survivors Germination of spores fDDm) cfu/ml in shake flask 100 0 100 0 - 100 107 + 200 105 + 300 102 + 200 106 + 300 106 + Table XIII Efficacy of Formulations A and B and of Caspan and Captan on Spores of Fusarium oxv.

Biocide Concentration (dditi) No. Survivors cfu/ml Germination of spores in shake flask None (Control) 1 x108 + - Formul. A 15 not measured + Formul. A 30 5x103 - Formul. A 45 0 - Formul. B 15 not measured + Formul. B 30 not measured + Formul. B 45 0 - Caspan 30 not measured + Caspan 45 not measured + Caspan 60 not measured + Captan 30 not measured + Captan 45 not measured + Captan 60 not measured + Table ΧίΥ Effect of Formulations A and B and of Caspan and Captan on Mvcellia of Fusarium oxv. so.

Concentration Dry Weight far) Growth on plates upon removal of biocide Glucose left in medium Biocide (DDm) Formul. A 200 0.13 (+-) 1 Formul. A 400 0.18 - 1 Formul. A 800 0.16 - 1 Formul. B 200 0.27 + 0.5 Formul. B 400 0.23 - 1 Formul. B 800 0.19 - 0.5 Captan 200 0.15 - 1-2 Captan 400 0.18 - 1-2 Captan 800 0.18 - 1-2 Caspan 200 0.48 + 0 Caspan 400 . 0.45 + 0 Caspan 800 0.30 + 1-2 None (Control) - 0.45 + 0

Claims (10)

1. A fumigator comprising a reservoir in which there is present a biocide which is 2. ,2-dibromo-nitrilopropionamide; 0 to 80 wt.% organic solvent; and 0 to 80 wt.% water.

2. A fumigator according to claim 1, biocide contains an organic solvent glycol. wherein the which is a

3. A fumigator according to claim 2, wherein the... 20 biocide contains a glycol selected from mono-,di- or· poly-propylene glycol.

4. A fumigator according to claim 3, wherein the biocide contains dipropylene glycol. 5. Sporistatic and/or a sporicidal in the treatment of crops. 23. Use of 2,2-dibromo-3-nitrilopropionamide to destroy or inhibit the growth of Fusarium oxysporum or

5. A fumigator according to any one of claims 1 to 4, ^wherein the biocide comprises up to 100 wt.% of 5 2,2-dibromo-3-nitrilopropionamide or a composition containing 2,2-dibromo-3-nitrilopropionamide in admixture with an organic solvent, water or a ' mixture of water with an organic solvent, the arrangement being such that in use the biocide is delivered from 10 the reservoir to the locus to be treated through gas as the transfer medium without any direct contact between the material present in the reservoir and the locus to be treated.

6. A fumigator according to claim 5, wherein the biocide comprises about 20 wt.% of 2,2-dibromo-nitrilopropionamide; about 60 wt.% organic solvent; and about 20 wt.% water.

7. A fumigator according to any preceding claim, further comprising means for heating the reservoir to bring about delivery of the biocide to the locus to be treated.

8. A method of treating crops infected or liable to be infected with microorganisms to inhibit or prevent growth of said microorganisms and/or germination of their spores, characterised in that the crop to be treated is fumigated with a biocide having the features set forth in any of claims 1 to 6.

9. A method according to claim 8, wherein the crop to be treated is potatoes or seed potatoes. 10. A method according to claim 8 or 9, wherein the microorganism is one or more fungi. 11. A method according to any one of claims 8 to 10, wherein the microorganism comprises Erwinia carotovora or Streptomyces scabies or Fusarium oxysporum or Verticillium dahliae or Rhizoctonia solani, or two or more of the said microorganisms . 12. Use as a sporicide or fungicide in the treatment of crops of 2,2-dibromo-3-nitrilopropionamide in an organic solvent, water or a mixture of water and an organic solvent. 13. Use according to claim 12, wherein the 2,2-dibromo-3-nitrilopropionamide is in a glycol as organic solvent. 14. Use according to claim 13, wherein the glycol is selected from mono-, di- and polypropylene glycol. 15. Use according to claim 14, wherein the glycol is di-propylene glycol. 16. Use as a sporicide or fungicide of a composition that contains: a) 5 to 25 wt.% 2,2-dibromo-3-nitrilopropionamide; b) 0 to 95 wt.% of water; and c) 0 to 95 wt.% of a glycol. 17. Use according to claim 16, wherein the glycol present in the composition is di-propylene glycol. 18. Use according to claim 17, wherein the composition contains: a) about 20 wt.% 2,2-dibromo-3-nitrilopropionamide; b) about 20 wt.% of water; and c) -about 60 wt.% of di-propylene glycol. 19. Use according to any of claims 11 to 18, wherein the composition is applied to potatoes or seed potatoes whenever fungi and/or bacteria and/or their spores are to be destroyed or their growth or germination inhibited. 20. Use according to claim 19, wherein the composition is applied by fumigation. 21. Use according to claim 19 or 20, wherein the crop to be treated is infected by Erwinia carotovora or Streptomyces scabies or Fusarium oxysporum or Verticillium dahliae or Rizoctonia solani, or by two or more of the said microorganisms. 22. Use of 2,2-dibromo-3-nitrilopropionamide as a

10. Verticillium dahliae on potatoes or seed potatoes when applied to the potatoes or seed potatoes at a dose sufficient to destroy the above organisms without inhibiting the growth of Trichoderma.