A Disease of Manuka Leptospermum scoparium Forst.
[Read before the Canterbury Branch, July 1, 1953; received by the Editor, September 8, 1953.]
A disease causing the death of Leptospermum scoparium Forst. in New Zealand is shown to be caused by a combination of an insect, Eriococcus manukae, and a fungus Capnodium walteri Sacc. This fungus has not been recorded previously in New Zealand.
In the final stages of the disease the host assumes a fire-blackened appearance duo to the covering of fungus. The shading effect on the leaves due to the cover of fungus was studied with a densitometer.
The fungus was isolated on a honey-dew decoction and maximum growth was obtained on Czapec-Dox agar with maltose as the carbohydrate source. Microspoies were produced on Czapec Dox Maltose Agar, Honey Agar, Malt Agar, Potato Dextrose Agar. No other spores were produced in culture. The morphology of the fungus is described and measurements given.
It has been possible to reproduce the disease on the host plant free from insects by spraying the plants with a 1% honey solution at regular intervals after inoculation with a culture of the fungus.
During the last decade a disease has appeared in New Zealand which is causing the death of manuka (Leptospermum scoparium Forst.). This disease is a new one in local records, and its origin remains undecided.
The death of L. scoparium over large areas has been greeted as a blessing by some farmers who look upon manuka as a weed associated with pasture deterioration and diminished stock-carrying capacity. On the other hand, there are those who contend that manuka has value as an aid in preventing soil erosion and in the provision of stock shelter.
Symptoms and Effects
A plant of L. scoparium infected with the disease shows no gross ill effects for approximately 2–3 months, although close inspection is likely to reveal the presence of scale insects. After six weeks to two months, examination shows that the main stems and most of the leaves in the centre of the plant have a thin covering of sooty mould. The insects are not confined to the stems, but are also found on the ventral and dorsal surfaces of the leaves, and indeed on all parts of the plant: they are most numerous on the stems, where the flaky type of bark affords them some measure of protection.
In the final stages of the disease the whole plant becomes covered with a black felt of mycelium, only a few leaves at the apices of the branches remaining unaffected (Fig. 1). After the death of the plant the fungus remains on the tree, giving it a fire-blackened appearance. The tree remains in this condition for some time (2–3 years), until the bark and fungus flakes off, leaving only the bleached branches.
Certain families of the Hemiptera whose members feed on the sap of plants are capable of excreting, without prior digestion, excess water and carbohydrates which they cannot metabolize. This fluid is voided via the anus, and is known as honey-dew. It contains a mixture of invert sugars and dextrin (Wrigglesworth, 1950). In the case of Eriococcus on manuka the insects living on the leaves and branches excrete this honey-dew, which is washed down the plant by dew and rain. Some of this solution is caught in the weft of the mycelium, where it is eventually used by the fungus as a source of energy. The honey-dew may be washed off manuka trees, and it is not uncommon to find plants and even the soil beneath diseased trees covered with the black fungus.
The Shading Effect of the Fungus on the Leaves
This experiment was designed to show that the fungus growing on the leaves caused a diminution in the amount of light available for photosynthesis.
Strips of adhesive cellophane tape were attached to microscope slides with the sticky side uppermost. A random sample of leaves covered with the fungus was taken and placed with the dorsal surfaces on the adhesive strip. A glass rod was gently rolled over the leaves, causing the fungus to adhere to the cellophane tape. The leaves were then removed and the tape covered with another layer of the cellophane tape.
The slides were examined with a densitometer (Western Electric Model 877). The machine was adjusted to give a zero reading when a portion of the slide with no fungus on it was placed beneath the head. It was found that 52% of the leaves tested allowed less than 50% transmission of light.
An experiment was set up to demonstrate the plant-fungus relationship. Disease-free plants growing in pots were sprayed with a 1% solution of honey in distilled water and then with a suspension of hyphal fragments prepared from a colony of Capnodium walteri growing on Potato Dextrose Agar. After inoculation the plants were held in a humid chamber for 48 hours.
The plants were kept in a glasshouse and sprayed at intervals of two weeks with 1% honey solution in distilled water. At the end of three months the plants possessed a light covering of black mycelium from which the fungus was isolated and grown in artificial culture. It proved to be Capnodium walteri. Control plants were inoculated with a suspension of hyphae, but subsequent sprayings were with distilled water only. The fungus could not be re-isolated from these plants.
Control of the Disease
The control of this disease on isolated specimens of Leptospermum scoparium presents no difficulty. It has been found that spraying once a year during the winter months with an oil emulsion is sufficient to kill the scale insects. This removes the source of nourishment of the fungus and prevents further growth. It has been found necessary to spray each year as the trees are readily reinfected with coccids. Naturally it would not be practicable to utilize this spraying technique in protecting manuka on isolated hill country where difficulties of access and topography would preclude the use of standard equipment.
Fig. 1.—Twigs taken from an infected manuka (Leptospermum scoparium) The two piece in the centre have been split to show the thickness of the sooty mould. (× 1.)
Fig. 2.—Section through a microsproangium showing the microspores being exuded through the ostiole. (× 500.)
Fig. 3.—A crushed perithecium showing an aseus and ascospores. (× 325.)
Fig. 4.—A crushed pyenidium with the pyenospores in situ. (× 350.)
Stems and leaves covered with the fungus were fixed in the Held with weak chrom-acetic fixative (Johansen, 1940). From this material serial sections were prepared, using alcohol for dehydration, and embedded in paraffin wax under reduced pressure. Several stains were tried, Pianese IIIB (Peacock, 1945) proving the most effective.
Examination of the sections showed that neither on the stem nor on the leaves did the hyphae produce haustorial organs of any sort, nor was the living tissue of L. scoparium penetrated in any way.
Isolation of the Fungus
Twigs 1–2 cms. long, covered with mycelium were washed for 8 hours in a suitable laboratory washing device. Surface sterilisation with the usual agents was found to be harmful to the fungus. After washing, one or two twigs were placed in a sterile test-tube with 3–5 ccs. of sterile water, and ground with a sterile rod. The resulting suspension of hyphal fragments was used to make dilution plates. It was found that the fungus could be isolated most readily on a decoction of L. scoparium twigs which were heavily infected with coccids and therefore contained a large quantity of honey-dew. The twigs were soaked in hot water for several hours. The resultant liquid was turbid with fragments of hyphae, and had a sweetish smell. This liquid was filtered, the filtrate solidified with 1.5% agar and sterilized at 101b pressure for 20 minutes. Dilution plates poured with this medium were examined microscopically with a low-power objective after incubation for 3 days at 24° C. Germinating hyphae well-separated from contaminants were transferred to tubes containing the same medium.
Several agar media were employed to determine the colony characteristics of the fungus. Czapek-Dox medium, made according to Thom and Raper (1945), was used with various carbohydrates—i.e., sucrose, dextrose, maltose, dextrin. Saboraud's Agar, Malt Agar and Potato Dextrose Agar according to Ainsworth and Bisby (1945) were also used.
Maximum growth was obtained on Czapek-Dox Agar with maltose of pH 6.8 at 24° C.
Aerial hyphae were produced on Prune Agar, Saboraud's Agar, Difco Lima Bean Agar and Czapek-Dox Agar. Microspores were produced abundantly on Czapek-Dox Maltose Agar, Honey Agar (5% honey, 2% agar), Malt Agar and Potato Dextrose Agar. On these media the colonies assumed a white slimy appearance due to the numerous microspores produced.
Microtome sections of cultures growing on agar were prepared in order to examine the colony structure. The colonies were found to consist of closely packed hyphae composed of doliform cells. Microsporangia (Fig. 2) were found scattered irregularly throughout the colony; their size and shape was irregular but tended from spherical to obclavate in form. The Avails are composed of two layers of isodiametric cells. A circular ostiole appears at the apex through which the microspores are exuded in large numbers embedded in a mucilaginous fluid. The microspores are oval in shape and thin-walled. They are usually without septa, but a few have one septum. Length varies from 2·7μ–5.5μ. (mean 4.0μ). and breadth 2.5μ–3.3μ (mean 2.8μ). No other spores were produced by the fungus in culture.
The description and measurements given below were made from fresh material mounted in glycerine jelly.
The hyphae (Fig. 3) were clear dark-brown in colour, darker in the older portions; and lighter towards the apices. Newly formed cells were almost hyaline with a greenish tinge; mature cells were doliform and much constricted at the septa while young hyaline cells showed no constriction at the septa. The cell wall was unsculptured, branches arising from any part of the hypha and in any plane. Numerous globules which stained with Sudan IV were present in the older cells. The cell measurements were 4.1μ–14μ long by 4.1μ by 15μ wide.
The flask-shaped pycnidia (Fig. 4) were borne partly imbedded in the mycelium, singly or in groups of two or three, both on the stems and leaves. The walls were made up of 3–5 layers of closely appressed cells. Pycnidia were observed at all seasons. The size was variable, 112μ–260μ in length, 45μ–100μ wide at the base and 19μ–32μ. wide at the neck. The pycnospores were fusiform, and often slightly curved, 21μ–36μ, by 3μ—4μ, golden-brown in colour and with usually 9 septa though these varied from 7 to 13.
Asci were produced in perithecia (Fig. 3) which are usually spherical, slightly flattened at the apex. The walls consisted of thick-walled cells of variable size. The assocarps measured 70μ–150μ long by 55μ–90μ wide.
Asci were obclavate and thin-walled. They contained 8 ascospores which were clavate, thick-walled and dark green-brown in colour. The number of septa varied between 2 and 5; the majority were found to have 3 septa. They measured 20μ–28μ long by 6μ–11μ wide.
The fungus described above agrees in all aspects, other than the breadth of the pycnospores, with the amended description of Capnodium walteri Sacc., given by Fraser (1935). Fraser gives the width of the pycnospores as 7μ-9μ: in my observations they were 3μ-4μ. However in an earlier description (Fisher, 1932) the average width is given as 5.5μ; it would not appear that this slight difference is great enough to warrant a new species.
The malady features a combination of insect and fungal parasites. The former belongs to the family Coccidae within the genus Eriococcus; The fungus Capnodium walteri Sacc. (Fraser, 1935), is a member of the Sooty Mould group and is nourished by honey-dew secreted by the insects which infest L. scoparium. This fungus has not been recorded previously in New Zealand. The disease is confined mainly to L. scoparium; L. ericoides, which is often found growing in L. scoparium stands, may be attacked but is rarely killed.
List of References
Ainsworth, G. C. and Bisby, G. R., 1945. A Dictionary of the Fungi. Imperial Mycological Institute, Kew.
Fisher, E. E., 1933. The Sooty Moulds of Some Australian Plants Proc. Roy. Soc. Victoria, XLV Pt. II (New Series) 171–202.
Fraser, L., 1935. An Investigation of the Sooty Moulds of New South Wales. IV. Proc. Linn. Soc. N.S.W., 60, 159–178.
Johansen, D. A., 1940. Plant Microtechnique. McGraw Hill Book Co., New York and London.
Thom, C. and Raper, K. B., 1945. A Manual of the Aspergilli. Williams and Wilkins & Co., Baltimore.
Wrigglesworth, V. B., 1950, The Principles of Insert Physiology, Methuen, London.