Researches on various kinds of fungi observed on Turkish opium, their action on the quality of opium and their removal with different sorts of fungicide

Abstract

I. INTRODUCTION

Details

Author: Hiket Eltutar, Mustafa Igneciler
Pages: 12 to 16
Creation Date: 1958/01/01

Researches on various kinds of fungi observed on Turkish opium, their action on the quality of opium and their removal with different sorts of fungicide

Dr. Hiket Eltutar
Mustafa Igneciler
Soil ProductsOffice of Turkey, Opium Section

I. INTRODUCTION

When cases of opium purchased from different districts of this country are opened for inspection the opium is sometimes found to be covered with mould.

It has been observed that the extent of mould is always greater on opium with a high moisture content. Mould is considered to be due to contamination of the opium while in the producer's possession. When the opium arrives in our warehouse in a contaminated condition it is observed that as a result of the process during which it is manipulated and pressed into the required export shape, mould spreads only over surfaces in contact with air.

Fungi are closely associated with moisture. The speed and extent of their growth depend on dampness and it would therefore be possible to avoid mould by maintaining a dry atmosphere permanently in the warehouse and preventing humidity. From a technical standpoint it would not, however, be convenient to eliminate humidity altogether in the warehouse; besides, this would not be possible, in our opinion, in a building situated near the sea.

We therefore contemplated using fungicides to protect the opium from mould. Part of the research undertaken for this purpose was completed in the Soil Products Office's laboratory; isolation of fungi was carried out in the Veterinary Bacteriological Institute at Pendik; identification was made in the Botanical Institute at the Istanbul University Faculty of Science (Prof. Dr. Heilbron); experiments on isolation of fungi, culture and tests on application of various preparations were performed under the supervision of Dr. Ibrahim Karaca, professor, the Chair of Plant Protection, Faculty of Agriculture of Ankara University.

II. MATERIALS AND METHOD

A . Materials

Opium used in the tests was obtained from various regions of Turkey and supplied by the Soil Products Office.

The following table shows the regions from which samples were obtained in order to study the action of fungus in relation to morphine content and morphine percentage.

One and a half kilos of each of the above samples drawn on 5 to 7 March 1958 were ground four times in the mincing machine and made homogeneous.

TABLE 1

Origin of samples, average morphine percentage and numbers of cases containing samples of 1953 crop

Origin
Morphine percentage
Serial numbers of cases
Erbaa
11. -
5823
Yalvac
11. -
5378
Bolvadin
11.50 3883
Emet
11.50 992
Zile
11.75 5441
Tavsanli
12. -
1443
Civril
12. -
5963
Bozkir
12.25 1553
Amasya
13.75 683
S. Karaagac
12. -
5072

The sample used in tests for fungicides was of the standard export type weighing 2 kilos. The preparations used in experiments were sold on the open market and were supplied by manufacturers to the Faculty of Agriculture and the Institute of Agricultural Control, Ankara.

Names of the fungicides used

Name
Manufacturer
Group according to active substance
Agresan GN
I.C.I.
Organic mercury (Tolymercuriacetate) Mercury 1%
Anticarie SD
Saint-Denis
Hexachlorbenzene
Arasan
Du Pont
Tetramethythiuramdisulphide
Ceresan T
Bayer
EthylmercurychlorideC 2N 5Hg C1
Cuprevit Ob 21
Bayer
50 % Copper
Dithane
 
 
Fermate
Du Pont
Dimethyldithiocarbamate de Fer
Netzschwefel
Schering
Sulphur 90%
Ortocide 50
Standard Oil Company U.S.A.
Combined Nitrogen and Chlorin
Perenox
I.C.I.
Copper 50%
Solbar
Bayer
Bariumpolysulphide
Surmesan
Koruma
 
Sulphur powder
Keciborlu
Sulphur
Verdesan
I.C.I.
Organic mercury

B. Method

The method used for determination of the action of fungus over morphine in opium was the lime-water method.

Czapek-Agar medium was used for reproduction of fungus. According to (1), components of the above medium are as follows:

Czapek-gar Medium

Water
1,000 cc
NaNo 3
3 g
K 2HPo 4
1 g
Mg.So 47H 2O
0.5 g
KC1
0.5 g
FeSO 47H 2O
0.01 g
Sugar
30 g
Agar
15 g

Inoculation of Aspergillus was effected in Czapek medium poured in Petri containers and the product was distributed by quantities of 25 mg for each Petri box. Then the Petri boxes were placed in a thermostatically controlled container at 30°C and were left to allow reproduction of fungus.

With regard to tests on opium, the opium was cut in shapes of cakes 0.5 cm square, so that they could be held in Petri boxes. These cakes were softened in water; then fungus was inoculated into them. Thereafter, the opium cakes were laid in the powder product and were completely covered with the powder and entirely hidden. Then they were placed in Petri boxes containing damp blotting paper. The Petri boxes were left in the thermostatically controlled container at 30°C. or at room temperature of 25-27°C.

III. VARIETIES OF FUNGI GROWING ON NATURAL OR MANIPULATED OPIUM

In this country, four kinds of disease are known to afflict poppy plants (Papaver somniferum L.) owing to such factors as climatic and soil conditions. Among these are cryptogamic parasites. However, available publications do not show the kinds of fungi attacking opium poppy. It has been observed here that opium in warehouses sometimes grows mouldy; mould factors always showing the same etiological and symptomatological properties were isolated at the Veterinary Bacteriological Institute at Pendik and were identified by Prof. Dr. Heilbron.

One of the two kinds of fungus thus identified was indicated as Aspergillus Niger. According to (1), it appears that this fungus belongs to the series Aspergillus Niger A. Awameri Nakazawa. The second kind was identified as Aspergillus minimus Wehmer. A. Minimus species are not widespread, and are found on rare occasions on dry leaves (2).

The aspergillus system

Class
Ascomycetes
Order
Plectascimene
Family
Aspergillacea
Species
Aspergillus

According to (3) No. 3 Aspergillus species include 119 kinds. Up to the present only Aspergillus Niger has been found in Turkey. Now As. Minimus has been discovered in opium and only two varieties out of 119 have been determined in this country.

Both varieties have been found in raw opium purchased from different regions, and fungi growing on it are probably due to contamination either in the fields or while it is in the producer's possession.

IV. ACTION OF FUNGUS ON MORPHINE

In order to ascertain whether fungus in opium has any action on the morphine content, samples of 1.5 kg each were drawn from opium produced by regions mentioned in part II of this article and mixed four or five times in the small mincing machine.

When results of analysis showed that the opium had been made thoroughly homogeneous, the samples were divided into two parts and placed in glass jars of one litre each.

The lids of ten jars were closed; the other ten were left open, so that the opium remained in contact with air. Moisture and morphine content were determined on the same day.

TABLE 2

Moisture and morphine percentage on samples made homogeneous on 9.III.1955

Origin
Moisture percentage
Morphine percentage
Erbaa
16.42 14.51
Yalvac
12.02 12.68
Bolvadin
16.81 13.41
Emet
13.16 12.96
Zile
15.47 12.18
Tavsanli
16.87 13.82
Civril
13.99 13.28
Bozkir
12.95 13.13
Amasya
17.55 14.69
S. Karaagac
15.48 12.80

No mouldiness appeared on opium contained in the jars which had been left open; the samples in closed jars were gradually covered with mould, which spread completely over the opium within thirty days. The samples were tested at times, and their morphine content checked.

Whenever more than one analysis was required, this was repeated three times, but no error beyond the permitted margin was found. In order to compare the findings, results of analyses were calculated on dry product basis.

TABLE 3

Results of tests made on different dates on samples in open jars (not mouldy) calculated on dry product basis

 

Dales of analyses

 
Regions
9.III.1955 Morphine percentage
2.V.1955 Morphine percentage
23.XI.1955 Morphine percentage
17.II.1958 Morphine percentage
Difference between first and last tests. Morphine %
Erbaa
17.36 17.60 17.56 17.52 0.16
Yalvac
14.41 14.14 13.93 14.20 0.21
Bolvadin
16.12 15.78 15.60 15.98 0.14
Emet
14.86 14.96 14.53 14.60 0.26
Zile
14.40 14.47 14.17 14.25 0.15
Tavsanli
16.62 16.64 16.29 16.35 0.27
Civril
15.44 15.05 15.53 15.45 0.01
Bozkir
15.08 14.87 15.19 15.10 0.02
Amasya
17.81 17.87 17.91 17.85 0.04
S. Karaagac
15.14 14.93 14.79 15.00 0.14

As will be seen in the above table, differences in morphine contents between the first and last analyses are as follows : Bozkir, Amasya, Civril : 0.01 - 0.04; Zile, Erbaa, S. Karaagac, Boldavin : 0.14 - 0.16; Yalvaç, Emet, Tavsanli : 0.21 - 0.27.

In conclusion of the foregoing it is understood that for the dry product at any rate, opium without mould shows no difference in its morphine content even at the end of a period of three years.

TABLE 4

Results of tests made on different dates on samples in closed jars (mouldy) calculated on dry product basis

Morphine percentage

 
Dates of analyses
Difference between first and last tests
Regions
9.1955
2.V.1955
23.XI.1955
17.II.1958
 
Erbaa
17.36 17.21 17.92 17.52 0.16
Yalvac
14.41 13.97 14.08 14.36 0.05
Bolvadin
16.12 15.54 16.18 16.10 0.02
Emet
14.86 14.17 14.84 14.86
-
Zile
14.40 13.98 13.97 14.30 0.10
Tavsanli
16.62 15.92 16.86 16.70 0.08
Civril
15.44 14.56 15.07 15.33 0.11
Bozkir
15.08 14.63 15.20 15.20 0.18
Amasya
17.81 17.82 17.66 18.08 0.27
S. Karaagac
15.14 15.02 15.23 15.19 0.05

It will be noted from the table above that morphine content determined on opium produced by Emet was the same on the first and the last analysis; differences between first and last tests for morphine content in opium from Boldavin, Yalvaç, S.Karaagaç, Tavsanli and Zile range between 0.02 and 0.10. As for Givril, Erbaa, Bozkir and Amasya opium, differences between the first and the last analysis for morphine content are from 0.11% to 0.27%.

It may be concluded from these data that mouldy opium in closed jars, as well as mouldless opium in open jars, did not show any considerable difference in morphine content at the end of a period of three years.

V. ELIMINATION OF FUNGI BY MEANS OF FUNGICIDES

Action of fungicides over fungi was at first tested in an artificial medium (Czapek), then in a natural medium (opium); and prolonged studies were made.

A. Tests on the products in an artificial medium

Products obtained from different firms were sprayed at the rate of 25 mg for each Petri box and laid homogeneously on Agar-Agar surface. As will be seen in the following table, some of the products remained inert, whereas others showed long-lasting action.

In the following table, sign ( - ) indicates inertia and (+) indicates beginning of growth of fungus.

This test was repeated four times and the average of days shown in the table was taken.

TABLE 5

Number of days elapsed before beginning of growth of fungus

 
DAYS
Name of product
1 2 3 4 5 6 7 8 9 10
...
60
Agresan GN
-
-
-
-
-
-
-
-
-
-
 
-
Anticarie
-
-
-
-
+
+
-
+
+
+
 
+
Arasan
-
-
-
-
-
-
+
+
+
+
 
+
Ceresan T
-
-
-
-
-
-
-
-
-
-
 
 
Dithane
-
-
-
-
-
-
+
+
+
+
 
+
Fermate
-
-
-
-
-
-
+
+
+
+
 
+
Cupravit
-
-
-
-
+
+
+
+
+
+
 
+
Netzschwefel
-
-
-
+
+
+
+
+
+
+
 
+
Orthocide 50
-
-
-
-
-
-
+
+
+
+
 
+
Solbar
-
-
-
+
+
+
+
+
+
+
 
+
Surmesan
-
-
-
+
+
+
+
+
+
+
 
+
Pelenox
-
-
-
-
+
+
+
+
+
+
 
+
Sulphur powder
-
-
-
+
+
+
+
+
+
+
 
+
Verdesan
-
-
-
-
-
-
-
-
-
-
 
-
Control
-
-
-
-
+
+
+
+
+
+
 
+

Upon examination of the results it was noted that Aspergillus Awomari appeared within four days after culture. Growth of fungus was not prevented by the products Anticarie, Cupravit, Netzschwefel, Solbar, Surmesan, Perenox and sulphur powder. As for Arasan, Dithane, Fermate and Orthocide : their action ceased at the end of a week, and fungus began to grow.

On the other hand, the products Agrosan GN, Ceresan and Verdesan prevented growth of fungus during sixty days.

Each product was used in equal quantities of 25 mg. Some were spraying products, doses of which did not of course, correspond to powder doses. Should these have proved efficient, we should have contemplated their application to opium. But - as shown in the above table - the results were entirely insufficient. With regard to the powder products, these were tried in lower density (10 mg for each Petri box), and growth of fungus did not start much sooner, but it developed with greater speed later on.

During these experiments, we did not look for the minimum active dose to be applied, but studied potency of products and duration of their action. We therefore did not find it necessary to use lower doses than 10 mg in our researches.

B. Application of fungicides on opium

As started in part II of this article, Aspergillus Niger was inoculated into opium which was cut in the shape of cakes. Then the cakes were completely covered with the product and placed in Petri boxes, where fungus developed in inverse proportion with the product's efficiency.

By comparing the following table with the previous one, it will be noted that the same products gave the same results in both experiments, so that satisfactory action of Agrosan GN, Arasan, Cerasan T., Orthocide 50 and Verdesan was confirmed. Tests were continued with these products.

TABLE 6

Number of days elapsed before beginning of growth of fungus on opium treated with various fungicides

 

DAYS

 

DAYS

Name of product
1 2 3 4 5 6 7 8 9 10
 
Name of product
1 2 3 4 5 6 7 8 9 10
 
Agrosan GN
-
-
-
-
-
-
-
-
-
-
*
Solbar
-
-
-
+
+
+
+
+
+
+
 
Anticarie
-
-
+
+
+
+
+
+
+
+
 
Surmesan
+
+
+
+
+
+
+
+
+
+
 
Ceresan T
-
-
-
-
-
-
-
-
-
-
**
Sulphur powder
-
+
+
+
+
+
+
+
+
+
 
Cupravit
-
-
-
-
-
+
+
+
+
+
 
Orthocide 50
-
-
-
-
-
-
-
-
-
-
***
Dithane
-
-
-
+
+
+
+
+
+
+
 
Verdesan
(Available quantity insufficient)
 
 
 
 
 
 
 
 
 
 
Fermate
-
- .
+
+
+
+
+
+
+
+
 
Arasan
-
-
-
-
-
-
-
-
-
+
 
Netzschwefel
-
-
+
+
+
+
+
+
+
+
 
Control
-
-
+
+
+
+
+
+
+
+
 
Perenox
-
-
-
-
-
+
+
+
+
+
 
 
 
 
 
 
 
 
 
 
 
 

Observations : * 11 days later in one case. ** 15 days later in one case. *** 5 days later in one case : 10 days later in another case.

Experiments on opium with each one of the above products were repeated five times with the method described and results were as follows :

TABLE 7

Number of days elapsed before beginning of growth of fungus on opium with use of fungicide products which seemed encouraging

 

DAYS

Name of product
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Agresan GN
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Arasan
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
5
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
Ceresan T.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Orthocide 50
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
+
+
+
-
+
Verdesan
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-

As will be seen from table 7, no fungus appeared on opium after use of Agresan GN, Ceresan T, Verdesan, and no sign of it was observed. However, with the use of Arasan and Orthocide 50, fungus began to grow on the eleventh day and spread over the medium.

The active substance of the above three products, action of which proved efficient, is mercury.

VI. CONCLUSIONS

  1. Fungi observed on raw and manipulated opium are Aspergillus Awomeri Nakazava and Aspergillus minimus Wehmer.

  2. No mould appeared on opium samples preserved in open jars.

  3. Opium samples preserved in closed jars grew progressively mouldy depending on moisture content and were completely covered with mould within a month's time.

  4. No marked difference was observed in three years’ time in morphine content of opium samples, either with or without mould.

  5. Fourteen kinds of product containing various active substances were tested against fungi in artificial and natural media.

  6. Results obtained both in artificial and natural media completely confirmed each other.

  7. Products containing mercury (Agresan GN, Ceresan T. and Verdesan) applied to opium in natural media prevented growth of fungus, thereby giving entirely satisfactory results.

VII. LITERATURE

  1. CHARLES, Thos. 1945, A Manual of the Aspergilli, Baltimore, 1 x + 373

  2. VEHMER, 1899, Aspergillus Minimus, Bot. Control, 80 : 449- 461.

  3. Centraalbureau voor 1947, Sehimmelcultures, List of Cultures. Baarn, Holland 145

  4. MIGULA, W., 1927, Kriptogamen - Flora von Deutschland, Deutsch-österreich und der Schweiz. BD. III. Pilze.

  5. SÖZEN HAYRI, 1950, Analitik Toksokoloji : Istanbul Universit esi.