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Trichothecium acridiorum (Trabut) comb.nov. on red locusts
[ 275 ] Trans. Br. mycol. Soc. 49 (2),275-288 (lg66) Printed in Great Britain
TRICHOTHECIUM ACRIDIORUM (TRABUT) COMB.NOV. ON RED LOCUSTS By M. F. MADE LIN Department of Botany, University of Bristol (With 4 Text-figures) Events associated with the original discovery of Botrytis acridiorum Trabut (= Fusarium acridiorum (Trabut) Brongniart & Delacroix) in 18g1 are reviewed, together with the biological features and taxonomic treatment of the fungus. This same organism has recently been found as a superficial parasite of red locusts (Nomadacris septemfasciata) in the laboratory. Its morphology, development and physical relationship with its host are described. It is proposed that the species be transferred to the genus Trichothecium Link ex Fries. Its similarity to Didymopsis locustanae Prinsloo is noted. The possibility that the presence of the fungus is secondary to physical injury is discussed. It is able to grow and sporulate on lipids from the locust's epicuticle, and viable spores have been found in the film of wax which becomes deposited on the inside of rearing cages from the bodies of captive locusts. The parasite appears to have little potentiality as an agent for artificial biological control.
HISTORICAL REVIEW
Discovery and nomenclature of the fungus The organism currently known as Fusarium acridiorum (Trabut) Brongniart & Delacroix was first seen on locusts (Schistocerca gregaria Forskal) in Algeria by Kunckel d'Herculais & Langlois in the spring of 18g!. Conditions near Algiers at that time appear to have been particularly suitable for the development of this fungus because on 23 May 18g1 it was again collected, this time by Prof. L. Trabut of the school of medicine in Algiers. On 8June 18g1 a telegram from Charles Brongniart (1891 a) announced to the Academic des Sciences in Paris that in Algeria he too had found locusts which had been killed by a fungus which he called a species of Botrytis. Trabut reported his discovery of the fungus to the Academic one week later, and described it as a new species, Botrytis acridiorum (Trabut, 1891 a). The following week, on 22 June 18g1, a paper by Kunckel & Langlois (1891) was read before the same body. They reported their original discovery of the fungus and described the organism. They said that it was a species close to Polyrhirium leptophyei Giard, which had been described 2 years earlier (Giard, 1889) from a collection on two male adults of another orthopterous insect, Leptopliyes punctatissima Bose. That same day Brongniart (1891 b) sent a second telegram to the Academic announcing that he had secured the Botrytis in culture, and followed this the next week (29June 1891) with an account of the way in which locusts in Algeria were being attacked by this organism for which he used Trabut's name (Brong18-2
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niart, 18g1 c). At that same meeting Giard (18g1 a) described Kunckel and Langlois' fungus, of which specimens had been sent to him on zo june 18gl, as a new species of a new genus, Lachnidium acridiorum. Trabut (I 8g I b) in later describing further details of the fungus and its action on locusts accepted Giard's new name. On 28 October 18g1 Brongniart and Delacroix (Brongniart, I 8g I d) proposed that the fungus be called Fusarium acridiorum (Trabut) Brongniart & Delacroix. Giard (I 8g2) was very critical of this transfer. The species underwent no further nomenclatural or taxonomic change until Wollenweber & Reinking (lg35) listed F. acridiorum as a facultative synonym of Fusarium solani (Martius) Appel & Wollenweber. This proposed synonymy was evidently not based on an examination of material of the locust fungus.
Biology of the fungus The Algerian locust parasite was described as entirely superficial on the cuticle, which it caused to darken (Trabut, 18g1 a; Brongniart, 18g1 c). Kunckel & Langlois (18gl) recorded that though superficial it was capable of occasionally deforming segments of the body to the extent of impeding egg-laying. Brongniart (I 8g I c) described the production of small, plump, brownish swellings at the junction of the dorsal and ventral abdominal sclerites, and like Giard (I 8g I a) recorded the presence of mycelium on the hind-feet as well as on the body. Ktmckel & Langlois (18gl) reported that it attacked locusts only at the end of their life-cycle, but Brongniart (18glc) recorded that in a considerable epidemic in Algeria in June 18g1 most of the many females died without laying eggs. Kunckel & Langlois (18gl) and Trabut (18g1 b) claimed that the disease was not very contagious. It was also slow to develop (Trabut, 18g1 b; Giard, 18g2). Giard (1892) stated that it became dangerous to the insect only when it invaded the tracheae and caused asphyxiation. Giard (I 8g I a) reported that the fungus occurred in two forms. A so-called Cladosporium-form produced a white powdery mass on the head, thorax, first abdominal segment and hind-feet of the affected locust, and formed small non- or I-septate spores; and a Fusarium-form, which was chiefly located on the underside of the fifth or sixth abdominal segments, formed a greyish down on which arose rather elongated, mostly curved, sometimes I-septate spores. In a later paper in which he interpreted the apparent variability of Lachnidium in terms of polymorphism, Giard (I 8g I b) suggested the alternative names Hormodendron-form and Fusisporium-form respectively for the two states. Giard was able to secure the Fusarium-form in artificial culture, but not the Cladosporium-form. Trabut (18g1 b) similarly observed the Fusarium-form in artificial cultures of the pathogen on gelatine-peptone medium, but was unable to find it on living diseased locusts. Giard (I 8g2) observed the production of thick-walled chlamydospores, generally in aggregates, in artificial culture of the Fusarium-form. He suspected that the spores of the fungus must have been scattered widely in numerous localities around Algiers. Because Trabut had observed that it was chiefly female locusts which were infected, and particularly those which had already oviposited once, Giard suspected that contamination of the
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insects might have occurred during oviposition in the soil, and that infection might have been assisted by the soft part of the integument becoming damaged during egg-laying. It was remarked by Giard in 1892 that it had not been proven that the fungus could also attack eggs. Marchionatto (1933) recorded a species of Fusarium, said to be allied to F. acridiorum, which covered the eggs of Schistocerca paranensis Burmeister with a white cottony growth. Akbar, Haque & Abbas (1958) similarly recorded a sparse white cottony mycelial growth on the eggs of Schistocerca gregaria which they said compared favourably with Saccardo's (1892) description of F. acridiorum. However, there appears to be no firm proof that the species of fungus which superficially parasitized adult locusts could also attack locust eggs. Though the fungus has been mentioned in literature several times since 1892, principally in compilations, few new facts have been added. MATERIAL AND METHODS
The author's attention was drawn by Dr M. Norris (Mrs O. W. Richards) of the Anti-Locust Research Centre, London, to the occurrence of brown lesions on the abdomina of adult red locusts (Nomadacris septemfasciata Serville) in laboratory stocks. These lesions, principally centred upon the ventral intersegmental folds, did not become very extensive, but the affected locusts generally failed to achieve sexual maturity. Some lesions bore a whitish material which consisted of fungal hyphae and small, ellipsoidal, sometimes I -septate spores. From a number of abdominal lesions cultures of similar fungi were isolated. The ventral abdominal lesions varied in aspect apparently according to their individual stage of development. Some were dull black, slightly raised, and sharply demarcated from the surrounding healthy integument. Others showed a macroscopically visible whitish granular zone at the centre which comprised the sporulating fungus. Some were rather more complex, showing a central dark spot surrounded by a translucent zone, a narrow white band, and a fuscous brown halo. Other lesions were centred upon the membranes joining the abdominal terga and sterna at the sides of the body, and on the articulating membranes at the limb bases. In addition, blackened and often fungoid lesions were common on the arolia and tarsal segments of the limbs (see Madelin, 1963, figure 2). These lesions yielded isolations of the same fungus as was obtained from the abdomen. Other fungoid sites included the antennae, the tips of palps, and surfaces of the head and compound eyes. Even when fungus was not detectable on abdominal lesions examined under a dissecting microscope, superficial mycelium could be seen after boiling excised pieces of cuticle in distilled water and then mounting in lactophenol with cotton blue. The mycelium revealed in this way varied from a delicate tracery to a rather compact layer, and sometimes bore occasional globose to ellipsoidal unicellular spores. Lesions on abdomina, feet and antennae were examined after embedding material in wax and cutting sections with a microtome. Material was fixed in Bouin's fixative at 60° C. for It hr., dehydrated in two changes of
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dioxan, in which it was left overnight, and then embedded, after one change of wax, in 52° M.P. paraffin wax with 0'5 % beeswax. The fungus was entirely superficial in all the lesions examined, lying closely applied to the integument amidst the microscopic spines and scale-like patterning on the cuticle (Fig. I). The same fungus was observed to develop after the death of a locust on parts of its head, abdomen and appendages. The cause of this particular insect's death is not known.
Fig, I, Transverse sections of: A, the membrane joining the tergum (t) and sternum (s) of an abdominal segment; and B, the tarsus of a hind-limb of Nomadacris septemfasciata, both showing the superficial location of the locust-fungus (f), (Magnification: A x 60; B, X34), OBSERVATIONS
Structure and development
ofthe fungus
The morphology of the fungus was difficult to discern on the host insect, where generally the superficial mycelium bore many spores of obscure origin. The white mycelium comprised septate, hyaline hyphae, 1-21£ wide, mostly c. I ,81£, bearing mainly subglobose to ellipsoidal unicellular spores which in samples from different lesions ranged from 3-51£ long and I '5-31£ wide, up to 3-71£ long and 2'5-3'51£ wide. Several pallid guttules or small vacuoles were sometimes visible. A rather small proportion of 2-celled spores was occasionally present. These were 5-7'51£ long and 3-3'51£ wide (Fig. 2A). The origin of the spores was possibly on lateral pegs on the hyphae and on short lateral conidiophores, but because the spores were naturally deciduous or readily dislodged, very few were seen attached to parent hyphae. The structure and development of the parasite were elucidated from studies of cultures, in particular, slide cultures, of nine isolates from different lesions. These were substantially alike in respect of their conidia, chlamydospores and arthrospores. On most media the colonies were white, sometimes becoming tinged with cream. They generally bore abundant but compact aerial mycelium, and were rather slow growing, e.g, 1-2 em. diam. after IO days at 20-25°. The vegetative mycelium was composed of fine, branching hyaline hyphae, 1-3'51£wide according to conditions of cultivation. Young hyphae were cylindrical, older ones sometimes slightly inflated with constricted
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septa. Anastomoses were frequent, in particular where hyphae were appressed to glass surfaces and in cultures on wax films (see below and Fig.
3G, H). Conidia were hyaline with smooth thin walls, and variable in size, shape and septation according to the conditions under which they were formed (Fig. 2 B-1). Unicellular conidia were generally ellipsoidal, sometimes subglobose, 4-6'5,u long, but occasionally more, and 2'5-3'5,u wide. Twocelled conidia were generally 7-20,u long and 2'5-6,u wide. Three- and
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Fig. 2. Trichothecium acridiorum. A, from colony on tarsus of fore-limb of specimen 1095; a piece of mycelium is also figured. B-1, From different cultures of isolates from several diseased locusts: B, from spec. 1095, on Bacteriological Nutrient Agar (BNA) plate; G-F, from spec. 1100, C from r-day slide culture on BNA, D from 37-day slant culture on BNA, E from s-day culture on broth of 0'5 % peptone-j-o-g % beef extract, F from 9-day culture on plate of 0'1 % peptone-l- 12 % gelatine; G-I from spec. 1094, G from 37-day slant culture on BNA, H from ro-day culture on malt agar, I from r-day slide culture on BNA.
four-celled conidia fell within the same range. Two-celled spores were commonly constricted at the septum. In spores with 2 or 3 septa, the constriction was most pronounced at that septum nearest the middle of the spore. The proximal cell of z-celled spores was generally larger than the distal one. Young cultures on media which favoured sporulation generally
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produced rather large conidia of which a high proportion were z-celled and a number 3- and 4-celled. Older cultures produced a greater proportion of unicellular conidia (Fig. 2D, G) as did cultures on films of the wax from the surface oflocust bodies (see below). The conidia originated at the apices of conidiophores. Though the latter varied much in size and form under different environmental conditions, the actual mode of conidium production remained basically unchanged.
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Fig. 3. Trichothecium acridiorum. Production of conidia. A-D, Conidiophores in slide cultures on BNA, seen in profile (hence not all septa are shown); A, isolate from spec. 1100, B from spec. 1095, C from spec. log4, and D from spec. IOg8. E, Two conidiophores from r s-day culture of isolate from spec. IOg4 on 0'1 % peptone+ 12% gelatine. F-K, Isolate from spec. IIOO; F, conidiophore beginning to form second conidium, from g-day culture on plate of o· I % peptone + 12% gelatine; G, anastomosing mycelium, with simple rather short conidiophores, in g-day cultures on plate of 0'1 % peptone+ 12 % gelatine; H, mycelium with short conidiophores from near edge of g-day slide culture on BNA; I, chain of arthrospores producing short conidiophores or lateral sporogenous pegs, from g-day slide culture on BNA; J, arthrospores producing globose conidia directly on g-day slide culture on BNA; K, conidium with two scars, from ro-day slant culture on malt agar. (Magnification: A-D, x 440; E-J, x 600; K, x 1350.)
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In slide cultures on Bacteriological Nutrient Agar (BNA) the conidiophores which arose from the medium were 10-1701£ long, simple or sparingly branched, sparsely septate or even non-septate if short, cylindrical, 1'5-31£ wide, and tapered near the tip to c. 11£ wide. Conidia were produced apically in almost the same manner in which they are formed by Yrichothecium roseum (Pers.) Link ex S. F. Gray (Ingold, 1956). The processes of sporogenesis are illustrated in Fig. 3. The first sign of the production of a spore was the appearance of a small globose swelling at the tip of the conidiophore which enlarged eventually to become somewhat rectangular in profile, though generally slightly wider below than above. At this stage a
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Fig. 4. Trichothecium acridiorum. Chlamydospores formed in culture on wax films. A, Isolate from spec. 1095; B, C from spec. 1089; D, E from spec. 1094. C also shows arthrospore production.
septum delimited the spore from the apex of the conidiophore which then began again to swell to form the second spore. The first was generally tipped to almost a right angle with the axis of the conidiophore by the gradual orientation of the second spore roughly in line with the conidiophore (Fig. 3 E, F). This process was repeated, forming a recurved basipetal chain of spores. No septate spore was ever seen attached directly to the tip of the conidiophore. It appears that not until a spore had been succeeded by at least one other did it develop a transverse septum. Second and even third septa sometimes grew across the spore as it lay in the chain. Detached conidia sometimes showed a scar of attachment 11£ across. Fig. 3 K illustrates a conidium which clearly shows this as well as the scar where the next older spore was joined. On older or lesswell nourished mycelia the conidiophores were generally short, being either single-celled tapering branches some r o-eojz Iong and 2'51£ wide at the base, or lateral non-septate tubular extensions of cells of vegetative hyphae, or merely peg-like lateral processes on such cells (Fig. 3 G, H, I). Conidiophores showing these high degrees of reduction generally produced unicellular conidia in the manner described above. Peg-like conidiophores commonly bore only a single conidium.
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Two other sorts of asexual spore have been seen, particularly in old cultures or on parts of the mycelium which had grown against glass surfaces. Arthrospores were formed by the rounding off and abstriction of short more or less rectangular segments of vegetative hyphae. These spores were rather thin-walled. In old parts of cultures the septa in the vegetative mycelium were closer together than in the young parts (e.g. 6-13,u apart, as cr. 33-55,u), presumably as a result of intercalation of new septa. These short segments sometimes became rather inflated and eventually separated as arthrospores. Some of the latter, while still attached to one another in the parent hypha or after separation, were seen to germinate to produce short conidiophores with terminal conidia (Fig. 3I,J). The third type of asexual spore was the chlamydospore. These were usually found in pairs but sometimes threes or fours, and if terminal matured in a basipetal sequence. They had smooth, only moderately thick walls (c. o'5,u), were golden-brown, and contained a cluster of approximately globular masses of refractive material. They ranged from 4 to 11,u diam. (Fig. 4A-E).
Comparison with the Algerian locust parasite Trabut (18g1 a) described Botrytis acridiorum as having short mycelium bearing on small branches groups of hyaline spores, globular or ellipsoidal or oblong and IO-15,u long, with shining granules. In the mass they formed white, fairly compact, farinose spots on segments ofthe abdomen of Schistocerca gregoria, especially on the folded membranes which join the segments. Trabut (18g1 b) pointed out that sections showed that the fungus was wholly superficial, confirming the finding of Kunckel & Langlois (18g1). Trabut referred to two sorts of spore: subglobose, unicellular and 6,u across; and elliptical, septate and 8-12,u long. This account of two sorts of spore also confirmed the report of Kunckel & Langlois, who described what was undoubtedly the same organism as having small ovoid spores about 6,u long in groups of two or three at the tips of hyphae, and larger oval spores, 9 by 5,u, divided by a septum which slightly constricted the spore. It also agreed with the report of Brongniart (189I c), who, like Trabut, Kunckel, and Langlois, had collected the fungus in the field. In describing Lachnidium acridiorum as a new genus and species, Giard (1891 a), who worked in Paris with material sent from Kunckel & Langlois in Algeria, described his so-called Cladosporium-form as similarly having ovoid unicellular spores c. 6,u long as well as I-septate, slightly constricted spores 8-12# long. However, he also described what he called the Fusarium-form, which produced a fairly long, greyish down on the abdomen and gave rise to spores which were generally curved, often septate, 12,u to at least 28# long, and in groups of I-6. He reported that he was unable to cultivate the Cladosporium-form, but was successful with the Fusarium-form which then produced even longer spores (25-3S,u), often with 3-4 septa, and which caused the culture medium rapidly to assume a yellow tint. Trabut (1891 b) commented that he had not encountered the Fusarium-form on live locusts, but instead had obtained it only in cultures which when old became slightly tinged with pink. Giard (1892) later reaffirmed that the Cladosporium-forms did not persist in his cultures which gave rise at once to the Fusarium-form.
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The latter produced swollen cells on sugary media and chlamydospores in old cultures. There is little doubt that the fungus described here by the present author is the same as that studied in Algeria by Kunckel and Langlois, Brongniart and Trabut, notwithstanding the fact that the host species oflocust is different. It agrees in the symptoms with which it is associated, its superficial position on the lesions on which it is found, and in its mycelial and conidial characteristics. To this extent it also fully agrees with Giard's (1891 a) description of the Cladosporium-form of his Lachnidium acridiorum. However, nothing has been seen of the Fusarium-form which Giard described. It is difficult to escape the conclusion that Giard's material, dispatched from Algeria to France, showed a secondary growth of a true Fusarium species, and that this or a similar Fusarium persistently contaminated his cultures. Trabut (1891 b) accepted Giard's interpretation of the relationship between the two 'forms' which he saw in his cultures, but nevertheless noted that he never encountered the Fusarium-form on live locusts. Though Brongniart and Delacroix (Brongniart, 1891 d) believed that Giard had probably united several different moulds under one name, they too thought that the Fusarium-form represented the eventual developmental state of the fungus first described by Trabut as B. acridiorum.
The taxonomic position
of the locust fungus
Trabut's (1891 a) disposition of his newly described species in the genus Botrytis is unsatisfactory. Lachnidium acridiorum Giard, which was explicitly established as a new genus and new species, was described from Kunckel & Langlois' material. It was not published as a transfer of Trabut's species even though Saccardo (1892) lists it as L. acridiorum (Trabut) Giard. L. acridiorum was probably based on imperfectly understood material comprising two distinct species of which one was undoubtedly B. acridiorum Trabut. L. acridiorum appears therefore to be a nomen confusum. Fusarium acridiorum (Trabut) Brongniart & Delacroix was proposed (Brongniart, 1891 d) in the belief that a Fusarium state was a stage in the fungus originally described by Trabut. In fact no Fusarium state was initially described by Trabut (1891 a) for B. acridiorum, and the new combination should have been cited as F. acridiorum (Giard) Brongniart & Delacroix being explicitly a transfer of Giard's fungus. The name F. acridiorum should also be disregarded, being based on a nomen confusum. The form and origin of the conidia of the locust fungus here studied and considered to be identical with Botrytis acridiorum Trabut are essentially the same as in Trichothecium roseum (Ingold, 1956), the type species of its genus, except that the smaller conidia of the locust parasite are not regularly produced to the right and left of the apex of the conidiophore. A search of the literature has failed to reveal a description of any species of Trichothecium or allied taxon with which the locust fungus might be synonymous. It is therefore proposed that Trabut's species be transferred to the genus Trichothecium Link ex Fries.
Transactions British Mycological Society Trichothecium acridiorum (Trabut) comb.nov.
Basionym: Botrytis acridiorum Trabut in C. r. hebd. Seanc. Acad. Sci., Paris, lIZ, 1384, 1891. A homonym of B. acridiorum may be a source of confusion. Vuillemin (191 I) referred to Botrytis acridiorum Brongniart & Delacroix, non Trabut. I have been unable to locate the origin of this name, but Vuillemin gives it as a synonym of B. delacroixii Saccardo. This is Saccardo's (1892) name for what Brongniart (189I d) referred to merely as a round-spored form of Botrytis. It is listed by MacLeod (1954) as a synonym of Beauveria bassiana (Balsamo) Vuillemin.
Comparison ofT. acridiorum with Didymopsis locustanae Prinsloo Prinsloo (1962) described a fungus which in South Africa attacked chiefly the intersegmental tissues of the brown locust, Locustana pardalina (Walker). She named it Didymopsis locustanae. Her description of this organism closely resembles the above account of T. acridiorum in respect of the appearance of the colony, and size and general form of the conidiophores and conidia, and the fact that both parasites are weak and do not penetrate the cuticle. The principal point of difference lies in the description of the aerogenous conidia of D. locustanae as being solitary while those of T. acridiorum are in basipetal chains. The early stages of sporogenesis illustrated by Prinsloo resemble comparable stages for T. acridiorum. It is not impossible that D.locustanae and T. acridiorum are the same fungus, for the fragile chains of spores of the latter are readily observed only in slide cultures. A developmental study of D. locustanae would clarify the relationship ofthese species.
Cultural characteristics of Trichothecium acridiorum Though isolates of T. acridiorum grew readily on a wide variety of nutrient media, sporulation was generally lacking or poor on media in common use in mycological laboratories. A systematic test of six isolates on a range of media revealed that except for one strain which remained sterile, all sporulated on BNA and also, to a generally lesser extent, on Sabouraud's Maltose Agar. Both of these media contain peptone. The restriction of the parasite to the surface of locust integument suggested the possibility that it was nourished by materials in the epicuticle and was unable to digest the procuticle. The epicuticle contains lipid material. An experiment was conducted in which the wax from six locusts (N. septemfasciata) was removed by running 1-2 ml. of ether over each and collecting the washings together. The solution oflipids was passed through a Whatman no. I filter-paper to remove suspended particles, and the faintly straw-coloured solution was then spotted repeatedly on to each of a series ofseven clean, polished microscope slides. Evaporation of the solvent left a bloom of wax. The slides were sterilized by storage overnight in propylene oxide vapour. The wax films were then each inoculated with a loopful of suspension of spores from an isolate of the locust parasite. When the droplets had dried the slides were incubated in a saturated atmosphere
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at 23° C. for 3 days, when mycelial growth could be seen at the site of the inoculum. The slides were then transferred to a series of vesselsin which the atmospheric humidity was controlled by saturated salt solution at 80, 84, 87, 88, 92, 97 and 100 % relative humidity ( R .H .) . After incubation for a further 26 days the slides were examined. At 100 % R.H. sparse mycelium had spread 8'5 mm. beyond the edge of the inoculum area and formed an abundance of conidia which were chiefly bicellular, though some were unicellular. Some older parts of the colony had fragmented into arthrospores. At 97 % R .H. the mycelium had advanced 4 mm. but though it had fragm ented into arthrospores it had formed no conidia. At 92 % R.H. and less the fungus had not spread beyond the original inoculum. It is evident that the ether-soluble materials from the locust epicuticleare able to sustain the fungus for vegetative growth and conidium production, and that the fungus though an ectoparasite is not especially tolerant of dry conditions. DISCUSSION
The relationship between Trichothecium acridiorum and the symptoms with which it is associated Though there seems little doubt that the dark lesions on the locusts studied were caused largely or even wholly by T. acridiorum, it has not been possible to prove this. Attempts to infect locusts artificially have not been conclusive because the fungus was apparently almost ubiquitous as a natural contaminant of the locust integument with the result that it always appeared in control populations if environmental conditions were favourable. Attempts to rid locusts of contamination failed. The addition of more spores to the body led to no perceptible increase in incidence of the lesions. The principal evidence for the role of the fungus as cause of the lesions is its constant association, but the possibility remains that the parasite secondarily invades parts of the integument which have already suffered harmperhaps slight physical injury-from some other agency. The parasite tends to become established most commonly on articulating surfaces, e.g. between abdominal sc1erites; but next to this develops most on surfaces which touch the locust's support, e.g. the ventral side of the abdomen and the tarsi. It can occur elsewhere, especially when conditions are humid. It is not clear whether this distribution is the result of mechanical abrasion assisting the establishment of the parasite, or the occurrence of regions of higher humidity, or exposure to sources of infection in the substrate. The following observations are relevant. Containers in which locusts are reared become smeared internally with a film of wax from the bodies of the locusts themselves. It was suspected that in the laboratory the fungus might persist in an active or quiescent state in this film. A clean microscope slide was therefore attached to the inside of such a container and left for 3 weeks. In this period it became smeared generally with a waxy material. It was removed and examined microscopically. Fungal spores resembling the locust fungus were present. The slide was then broken in two and the halves separately placed face down upon malt-agar plates for 3 days at 25°, in which period mould and yeast colonies developed under and around them. Subcultures were prepared
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and several pure cultures of T. acridiorum were obtained. Another slide left for 2 months in a cage containing locusts similarly showed spores morphologically like T. acridiorum in the covering wax film. Except for one spore with a short germ-tube, none had germinated. These observations indicate that though T. acridiorum survived in the wax film which came to line the rearing chamber, it did not actively grow there under the usual environmental conditions. The relationship of the fungus to mechanical injury is not clear, for experiments to investigate this were inconclusive. Locusts are apt to inflict injuries on one another or even on themselves, principally with the tibial spines of the hind limbs, particularly if rather crowded. Abrasion or even puncture by such spines might assist in establishing the fungus, particularly in the lateral folds between the abdominal sclerites. The latter are also sites where the microclimate by being more humid is likely to favour the fungus's growth. It is not at present possible to express an opinion on the relative importance ofinjury and humidity. Apart from wounding, the limbs might also serve as means of transferring spores from the substrate, or from colonies growing among the tarsal pads, to the sides of the abdomen. Although infected locusts died in the laboratory, their death generally followed late upon the appearance of fungoid lesions, and came after a period of diminishing vigour. Owing to the above-mentioned difficulties encountered in conducting infection tests, it has not been possible to decide whether the fungus was the cause of the weakened condition or whether it developed best on locusts ailing from some other cause. It was nevertheless clear that the strictly superficial parasite never caused acute fatal disease. This finding agrees with those of Kunckel & Langlois (189 I ), Trabut (1891 b) and Giard (1892), who refer to the diseased condition as benign. The fungus was found only on adult locusts. No infected nymphs were seen. Possibly the duration of the nymphal instars is too brief to allow the fungus to become established before it is shed with the larval skin at the next moult.
lrifection cycles It is possible that the fungus persists between parasitic phases on laboratory stocks oflocusts in the wax which smears the insides of cages. Rigorous cleaning of the insides of cages with detergent between their use for successive populations oflocusts appeared to reduce the incidence of the disease, but no quantitative data were collected. In so far as the fungus developed well on locusts held under abnormally humid conditions resulting from inadequate ventilation, it seems advisable to aerate laboratory culture chambers adequately if infection is to be minimized. Giard (1892) tentatively ascribed a role in the natural life-cycle of the parasite to the chlamydospores which he saw in his cultures of the Fusarium form of his organism. There is doubt as to which fungus the chlamydospores he saw really corresponded, but the fact that T. acridiorum here formed chlamydospores in culture allows that Giard's suggestion may be correct. In view of the doubt about Giard's Fusarium-form it is unlikely that the genuine Fusarium species reported on eggs of locusts by Marchionatto
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(1933) and Akbar et al. (1958) could have been T. acridiorum. There is still no proof that this parasite of adult locusts also attacks locust eggs. Significance if Trichothecium acridiorumfor biological control Of the workers who were concerned with the fungus in 18g1 when first it was notic ed, Brongniart considered it held high promise as a biological control agent. On th e other hand Kunckel & Langlois (1891) said that it appeared impossible to base hopes of destroying the locusts on the artificial development of this fungus. Trabut (18g1 b) expressed the view that it was insufficiently harmful to be really useful in control. Giard (1892) had similar reservations based on its mild nature, the difficulty in its transmission, and its attack in the field chiefly on spent locusts. The pot entiality of the fungus for biological control of locusts was the subject of a lively public controversy in 18g1 and early 18g2 between Brongniart and Giard and in which the popular press also participated (see Giard, 18g2, for references). The fact that more fungi th an T. acridiorum may have been involved in Algeria (see Brongniart, 1 8g 1 d) may explain some of the conflicting views. There are nevertheless no convincing reports of successful control oflocusts with T. acridiorum. It is reasonable to assume from a review of the relevant literature and from the experience of this present investigation that T. acridiorum holds little pot ential for artificial biological control. I am grateful to Dr M. Norris and other workers at th e Anti-Locust Research Centre, London, for supplying me with infected and healthy locusts. REFERENCES
AKBAR, K. , HAQ.UE, H. & AnBAS, H . M. (1958). Fusarium acridiorum, a parasite of desert locust. Pl. Prot. Bull. FAD, 6, 59. BRONGNIART, C. (1891a ). Les criquets en Algerie. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 131 8- 13 20 . BRONGNIART, C. (189 I b). Telegraph message, dated 19June 1891. C.r. hebd. Seanc. Acad. sa., Paris, 112, 1478. BRONGNIART, C. (189Ic). Le cryptogame des criquets pelerins. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 1494-1496. BRONGNIART, C. (189 1d). Les champignons parasites observes sur les criquets pelerins en Algerie (Acridium peregrinum Oliv .). Bull. Seanc. Soc. natn. Agric. Fr. 51, 627-636. GIARD, A. (1889). Sur quelques typ es remarquables de champignons entomophytes. Bull. scient. Fr. Belg. 20, 197-224. GIARD, A. (189 I a). Sur les Cladospori ees entomophytes, nouveau groupe de champignons parasites des insectes. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 1518-152 I. GIARD, A. (189 I b). Sur Ie champignon parasite des criquets pelerins (Lachnidium acridiorum Gd.). C.r. hebd. Seanc. Acad. Sci., Paris, 113,813-816. GIARD, A. (1892). Nouvelles etudes sur Ie Lachnidium acridiorum, Gd. champignon parasite du criquet pelerin, Revue gen. Bot. 4, 449-461. INGOLD, C. T. (1956) . The conidial apparatus of Trichothecium roseum. Trans. Br. mycol. Soc. 39, 460-464. KUNCKEL D'HERCULAIS, J. & LANGLOIS, C. (1891). Les champignons parasites des Acridiens. C.r. hebd. Sianc. Acad. Sci., Paris, 112, 1465-1468. MAcLEOD, D. M. (1954). Investigations in the genera Beauveria Yuill. and Tritirachium Limber. Can. ]. Bot. 32, 8 18-890. MADELIN, M. F. (1963). Diseases caused by hyphomycetous fungi. Insect pathology, an advanced treatise, vol. II, pp. 233-2 71. Ed. E. A. Steinhaus. New York and London: Academic Pr ess.
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MARCHIONATTO,]. B. (lg33). Parasites vegetales de la langosta. Boln Minist. Agric., B.
Aires, 34, 227-246. PRINSLOO, H. E. (I g62). Didymopsis locustanae, a new species of fungus on the cuticle of the brown locust. S. Afr. ]. agric. Sci. 5, 23-26. SACCARDO, P. A. (r892). Svlloge fungorum, IO, 964 pp. TRABUT, L. (18gla). Sur une maladie cryptogamique du criquet pelerin (Acridium pere-
grinum). C.r. hebd. Slane. Acad. Sci., Paris, U2, 1383-1384. TRABUT, L. (189 I b). Les champignons parasites du criquet pelerin, Revue gin. Bot. 3,
4°1-4°5·
VUILLEMIN, P. (1911). Les Isaria de la famille des Verticilliacees (Spicaria et Gibellula).
Bull. Soc. mycol. Fr. 27, 75-82. WOLLENWEBER, H. W. & REINKING, O. A. (1935). Die Fusarien, ihre Beschreibung, Schadwirkung und Bekdmpfung, 355 pp. Berlin: Paul Parey.
(Accepted for publication
20
July 1965)
TRICHOTHECIUM ACRIDIORUM (TRABUT) COMB.NOV. ON RED LOCUSTS By M. F. MADE LIN Department of Botany, University of Bristol (With 4 Text-figures) Events associated with the original discovery of Botrytis acridiorum Trabut (= Fusarium acridiorum (Trabut) Brongniart & Delacroix) in 18g1 are reviewed, together with the biological features and taxonomic treatment of the fungus. This same organism has recently been found as a superficial parasite of red locusts (Nomadacris septemfasciata) in the laboratory. Its morphology, development and physical relationship with its host are described. It is proposed that the species be transferred to the genus Trichothecium Link ex Fries. Its similarity to Didymopsis locustanae Prinsloo is noted. The possibility that the presence of the fungus is secondary to physical injury is discussed. It is able to grow and sporulate on lipids from the locust's epicuticle, and viable spores have been found in the film of wax which becomes deposited on the inside of rearing cages from the bodies of captive locusts. The parasite appears to have little potentiality as an agent for artificial biological control.
HISTORICAL REVIEW
Discovery and nomenclature of the fungus The organism currently known as Fusarium acridiorum (Trabut) Brongniart & Delacroix was first seen on locusts (Schistocerca gregaria Forskal) in Algeria by Kunckel d'Herculais & Langlois in the spring of 18g!. Conditions near Algiers at that time appear to have been particularly suitable for the development of this fungus because on 23 May 18g1 it was again collected, this time by Prof. L. Trabut of the school of medicine in Algiers. On 8June 18g1 a telegram from Charles Brongniart (1891 a) announced to the Academic des Sciences in Paris that in Algeria he too had found locusts which had been killed by a fungus which he called a species of Botrytis. Trabut reported his discovery of the fungus to the Academic one week later, and described it as a new species, Botrytis acridiorum (Trabut, 1891 a). The following week, on 22 June 18g1, a paper by Kunckel & Langlois (1891) was read before the same body. They reported their original discovery of the fungus and described the organism. They said that it was a species close to Polyrhirium leptophyei Giard, which had been described 2 years earlier (Giard, 1889) from a collection on two male adults of another orthopterous insect, Leptopliyes punctatissima Bose. That same day Brongniart (1891 b) sent a second telegram to the Academic announcing that he had secured the Botrytis in culture, and followed this the next week (29June 1891) with an account of the way in which locusts in Algeria were being attacked by this organism for which he used Trabut's name (Brong18-2
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niart, 18g1 c). At that same meeting Giard (18g1 a) described Kunckel and Langlois' fungus, of which specimens had been sent to him on zo june 18gl, as a new species of a new genus, Lachnidium acridiorum. Trabut (I 8g I b) in later describing further details of the fungus and its action on locusts accepted Giard's new name. On 28 October 18g1 Brongniart and Delacroix (Brongniart, I 8g I d) proposed that the fungus be called Fusarium acridiorum (Trabut) Brongniart & Delacroix. Giard (I 8g2) was very critical of this transfer. The species underwent no further nomenclatural or taxonomic change until Wollenweber & Reinking (lg35) listed F. acridiorum as a facultative synonym of Fusarium solani (Martius) Appel & Wollenweber. This proposed synonymy was evidently not based on an examination of material of the locust fungus.
Biology of the fungus The Algerian locust parasite was described as entirely superficial on the cuticle, which it caused to darken (Trabut, 18g1 a; Brongniart, 18g1 c). Kunckel & Langlois (18gl) recorded that though superficial it was capable of occasionally deforming segments of the body to the extent of impeding egg-laying. Brongniart (I 8g I c) described the production of small, plump, brownish swellings at the junction of the dorsal and ventral abdominal sclerites, and like Giard (I 8g I a) recorded the presence of mycelium on the hind-feet as well as on the body. Ktmckel & Langlois (18gl) reported that it attacked locusts only at the end of their life-cycle, but Brongniart (18glc) recorded that in a considerable epidemic in Algeria in June 18g1 most of the many females died without laying eggs. Kunckel & Langlois (18gl) and Trabut (18g1 b) claimed that the disease was not very contagious. It was also slow to develop (Trabut, 18g1 b; Giard, 18g2). Giard (1892) stated that it became dangerous to the insect only when it invaded the tracheae and caused asphyxiation. Giard (I 8g I a) reported that the fungus occurred in two forms. A so-called Cladosporium-form produced a white powdery mass on the head, thorax, first abdominal segment and hind-feet of the affected locust, and formed small non- or I-septate spores; and a Fusarium-form, which was chiefly located on the underside of the fifth or sixth abdominal segments, formed a greyish down on which arose rather elongated, mostly curved, sometimes I-septate spores. In a later paper in which he interpreted the apparent variability of Lachnidium in terms of polymorphism, Giard (I 8g I b) suggested the alternative names Hormodendron-form and Fusisporium-form respectively for the two states. Giard was able to secure the Fusarium-form in artificial culture, but not the Cladosporium-form. Trabut (18g1 b) similarly observed the Fusarium-form in artificial cultures of the pathogen on gelatine-peptone medium, but was unable to find it on living diseased locusts. Giard (I 8g2) observed the production of thick-walled chlamydospores, generally in aggregates, in artificial culture of the Fusarium-form. He suspected that the spores of the fungus must have been scattered widely in numerous localities around Algiers. Because Trabut had observed that it was chiefly female locusts which were infected, and particularly those which had already oviposited once, Giard suspected that contamination of the
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insects might have occurred during oviposition in the soil, and that infection might have been assisted by the soft part of the integument becoming damaged during egg-laying. It was remarked by Giard in 1892 that it had not been proven that the fungus could also attack eggs. Marchionatto (1933) recorded a species of Fusarium, said to be allied to F. acridiorum, which covered the eggs of Schistocerca paranensis Burmeister with a white cottony growth. Akbar, Haque & Abbas (1958) similarly recorded a sparse white cottony mycelial growth on the eggs of Schistocerca gregaria which they said compared favourably with Saccardo's (1892) description of F. acridiorum. However, there appears to be no firm proof that the species of fungus which superficially parasitized adult locusts could also attack locust eggs. Though the fungus has been mentioned in literature several times since 1892, principally in compilations, few new facts have been added. MATERIAL AND METHODS
The author's attention was drawn by Dr M. Norris (Mrs O. W. Richards) of the Anti-Locust Research Centre, London, to the occurrence of brown lesions on the abdomina of adult red locusts (Nomadacris septemfasciata Serville) in laboratory stocks. These lesions, principally centred upon the ventral intersegmental folds, did not become very extensive, but the affected locusts generally failed to achieve sexual maturity. Some lesions bore a whitish material which consisted of fungal hyphae and small, ellipsoidal, sometimes I -septate spores. From a number of abdominal lesions cultures of similar fungi were isolated. The ventral abdominal lesions varied in aspect apparently according to their individual stage of development. Some were dull black, slightly raised, and sharply demarcated from the surrounding healthy integument. Others showed a macroscopically visible whitish granular zone at the centre which comprised the sporulating fungus. Some were rather more complex, showing a central dark spot surrounded by a translucent zone, a narrow white band, and a fuscous brown halo. Other lesions were centred upon the membranes joining the abdominal terga and sterna at the sides of the body, and on the articulating membranes at the limb bases. In addition, blackened and often fungoid lesions were common on the arolia and tarsal segments of the limbs (see Madelin, 1963, figure 2). These lesions yielded isolations of the same fungus as was obtained from the abdomen. Other fungoid sites included the antennae, the tips of palps, and surfaces of the head and compound eyes. Even when fungus was not detectable on abdominal lesions examined under a dissecting microscope, superficial mycelium could be seen after boiling excised pieces of cuticle in distilled water and then mounting in lactophenol with cotton blue. The mycelium revealed in this way varied from a delicate tracery to a rather compact layer, and sometimes bore occasional globose to ellipsoidal unicellular spores. Lesions on abdomina, feet and antennae were examined after embedding material in wax and cutting sections with a microtome. Material was fixed in Bouin's fixative at 60° C. for It hr., dehydrated in two changes of
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dioxan, in which it was left overnight, and then embedded, after one change of wax, in 52° M.P. paraffin wax with 0'5 % beeswax. The fungus was entirely superficial in all the lesions examined, lying closely applied to the integument amidst the microscopic spines and scale-like patterning on the cuticle (Fig. I). The same fungus was observed to develop after the death of a locust on parts of its head, abdomen and appendages. The cause of this particular insect's death is not known.
Fig, I, Transverse sections of: A, the membrane joining the tergum (t) and sternum (s) of an abdominal segment; and B, the tarsus of a hind-limb of Nomadacris septemfasciata, both showing the superficial location of the locust-fungus (f), (Magnification: A x 60; B, X34), OBSERVATIONS
Structure and development
ofthe fungus
The morphology of the fungus was difficult to discern on the host insect, where generally the superficial mycelium bore many spores of obscure origin. The white mycelium comprised septate, hyaline hyphae, 1-21£ wide, mostly c. I ,81£, bearing mainly subglobose to ellipsoidal unicellular spores which in samples from different lesions ranged from 3-51£ long and I '5-31£ wide, up to 3-71£ long and 2'5-3'51£ wide. Several pallid guttules or small vacuoles were sometimes visible. A rather small proportion of 2-celled spores was occasionally present. These were 5-7'51£ long and 3-3'51£ wide (Fig. 2A). The origin of the spores was possibly on lateral pegs on the hyphae and on short lateral conidiophores, but because the spores were naturally deciduous or readily dislodged, very few were seen attached to parent hyphae. The structure and development of the parasite were elucidated from studies of cultures, in particular, slide cultures, of nine isolates from different lesions. These were substantially alike in respect of their conidia, chlamydospores and arthrospores. On most media the colonies were white, sometimes becoming tinged with cream. They generally bore abundant but compact aerial mycelium, and were rather slow growing, e.g, 1-2 em. diam. after IO days at 20-25°. The vegetative mycelium was composed of fine, branching hyaline hyphae, 1-3'51£wide according to conditions of cultivation. Young hyphae were cylindrical, older ones sometimes slightly inflated with constricted
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septa. Anastomoses were frequent, in particular where hyphae were appressed to glass surfaces and in cultures on wax films (see below and Fig.
3G, H). Conidia were hyaline with smooth thin walls, and variable in size, shape and septation according to the conditions under which they were formed (Fig. 2 B-1). Unicellular conidia were generally ellipsoidal, sometimes subglobose, 4-6'5,u long, but occasionally more, and 2'5-3'5,u wide. Twocelled conidia were generally 7-20,u long and 2'5-6,u wide. Three- and
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Fig. 2. Trichothecium acridiorum. A, from colony on tarsus of fore-limb of specimen 1095; a piece of mycelium is also figured. B-1, From different cultures of isolates from several diseased locusts: B, from spec. 1095, on Bacteriological Nutrient Agar (BNA) plate; G-F, from spec. 1100, C from r-day slide culture on BNA, D from 37-day slant culture on BNA, E from s-day culture on broth of 0'5 % peptone-j-o-g % beef extract, F from 9-day culture on plate of 0'1 % peptone-l- 12 % gelatine; G-I from spec. 1094, G from 37-day slant culture on BNA, H from ro-day culture on malt agar, I from r-day slide culture on BNA.
four-celled conidia fell within the same range. Two-celled spores were commonly constricted at the septum. In spores with 2 or 3 septa, the constriction was most pronounced at that septum nearest the middle of the spore. The proximal cell of z-celled spores was generally larger than the distal one. Young cultures on media which favoured sporulation generally
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produced rather large conidia of which a high proportion were z-celled and a number 3- and 4-celled. Older cultures produced a greater proportion of unicellular conidia (Fig. 2D, G) as did cultures on films of the wax from the surface oflocust bodies (see below). The conidia originated at the apices of conidiophores. Though the latter varied much in size and form under different environmental conditions, the actual mode of conidium production remained basically unchanged.
F
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H
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Fig. 3. Trichothecium acridiorum. Production of conidia. A-D, Conidiophores in slide cultures on BNA, seen in profile (hence not all septa are shown); A, isolate from spec. 1100, B from spec. 1095, C from spec. log4, and D from spec. IOg8. E, Two conidiophores from r s-day culture of isolate from spec. IOg4 on 0'1 % peptone+ 12% gelatine. F-K, Isolate from spec. IIOO; F, conidiophore beginning to form second conidium, from g-day culture on plate of o· I % peptone + 12% gelatine; G, anastomosing mycelium, with simple rather short conidiophores, in g-day cultures on plate of 0'1 % peptone+ 12 % gelatine; H, mycelium with short conidiophores from near edge of g-day slide culture on BNA; I, chain of arthrospores producing short conidiophores or lateral sporogenous pegs, from g-day slide culture on BNA; J, arthrospores producing globose conidia directly on g-day slide culture on BNA; K, conidium with two scars, from ro-day slant culture on malt agar. (Magnification: A-D, x 440; E-J, x 600; K, x 1350.)
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In slide cultures on Bacteriological Nutrient Agar (BNA) the conidiophores which arose from the medium were 10-1701£ long, simple or sparingly branched, sparsely septate or even non-septate if short, cylindrical, 1'5-31£ wide, and tapered near the tip to c. 11£ wide. Conidia were produced apically in almost the same manner in which they are formed by Yrichothecium roseum (Pers.) Link ex S. F. Gray (Ingold, 1956). The processes of sporogenesis are illustrated in Fig. 3. The first sign of the production of a spore was the appearance of a small globose swelling at the tip of the conidiophore which enlarged eventually to become somewhat rectangular in profile, though generally slightly wider below than above. At this stage a
~DO B
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Fig. 4. Trichothecium acridiorum. Chlamydospores formed in culture on wax films. A, Isolate from spec. 1095; B, C from spec. 1089; D, E from spec. 1094. C also shows arthrospore production.
septum delimited the spore from the apex of the conidiophore which then began again to swell to form the second spore. The first was generally tipped to almost a right angle with the axis of the conidiophore by the gradual orientation of the second spore roughly in line with the conidiophore (Fig. 3 E, F). This process was repeated, forming a recurved basipetal chain of spores. No septate spore was ever seen attached directly to the tip of the conidiophore. It appears that not until a spore had been succeeded by at least one other did it develop a transverse septum. Second and even third septa sometimes grew across the spore as it lay in the chain. Detached conidia sometimes showed a scar of attachment 11£ across. Fig. 3 K illustrates a conidium which clearly shows this as well as the scar where the next older spore was joined. On older or lesswell nourished mycelia the conidiophores were generally short, being either single-celled tapering branches some r o-eojz Iong and 2'51£ wide at the base, or lateral non-septate tubular extensions of cells of vegetative hyphae, or merely peg-like lateral processes on such cells (Fig. 3 G, H, I). Conidiophores showing these high degrees of reduction generally produced unicellular conidia in the manner described above. Peg-like conidiophores commonly bore only a single conidium.
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Two other sorts of asexual spore have been seen, particularly in old cultures or on parts of the mycelium which had grown against glass surfaces. Arthrospores were formed by the rounding off and abstriction of short more or less rectangular segments of vegetative hyphae. These spores were rather thin-walled. In old parts of cultures the septa in the vegetative mycelium were closer together than in the young parts (e.g. 6-13,u apart, as cr. 33-55,u), presumably as a result of intercalation of new septa. These short segments sometimes became rather inflated and eventually separated as arthrospores. Some of the latter, while still attached to one another in the parent hypha or after separation, were seen to germinate to produce short conidiophores with terminal conidia (Fig. 3I,J). The third type of asexual spore was the chlamydospore. These were usually found in pairs but sometimes threes or fours, and if terminal matured in a basipetal sequence. They had smooth, only moderately thick walls (c. o'5,u), were golden-brown, and contained a cluster of approximately globular masses of refractive material. They ranged from 4 to 11,u diam. (Fig. 4A-E).
Comparison with the Algerian locust parasite Trabut (18g1 a) described Botrytis acridiorum as having short mycelium bearing on small branches groups of hyaline spores, globular or ellipsoidal or oblong and IO-15,u long, with shining granules. In the mass they formed white, fairly compact, farinose spots on segments ofthe abdomen of Schistocerca gregoria, especially on the folded membranes which join the segments. Trabut (18g1 b) pointed out that sections showed that the fungus was wholly superficial, confirming the finding of Kunckel & Langlois (18g1). Trabut referred to two sorts of spore: subglobose, unicellular and 6,u across; and elliptical, septate and 8-12,u long. This account of two sorts of spore also confirmed the report of Kunckel & Langlois, who described what was undoubtedly the same organism as having small ovoid spores about 6,u long in groups of two or three at the tips of hyphae, and larger oval spores, 9 by 5,u, divided by a septum which slightly constricted the spore. It also agreed with the report of Brongniart (189I c), who, like Trabut, Kunckel, and Langlois, had collected the fungus in the field. In describing Lachnidium acridiorum as a new genus and species, Giard (1891 a), who worked in Paris with material sent from Kunckel & Langlois in Algeria, described his so-called Cladosporium-form as similarly having ovoid unicellular spores c. 6,u long as well as I-septate, slightly constricted spores 8-12# long. However, he also described what he called the Fusarium-form, which produced a fairly long, greyish down on the abdomen and gave rise to spores which were generally curved, often septate, 12,u to at least 28# long, and in groups of I-6. He reported that he was unable to cultivate the Cladosporium-form, but was successful with the Fusarium-form which then produced even longer spores (25-3S,u), often with 3-4 septa, and which caused the culture medium rapidly to assume a yellow tint. Trabut (1891 b) commented that he had not encountered the Fusarium-form on live locusts, but instead had obtained it only in cultures which when old became slightly tinged with pink. Giard (1892) later reaffirmed that the Cladosporium-forms did not persist in his cultures which gave rise at once to the Fusarium-form.
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The latter produced swollen cells on sugary media and chlamydospores in old cultures. There is little doubt that the fungus described here by the present author is the same as that studied in Algeria by Kunckel and Langlois, Brongniart and Trabut, notwithstanding the fact that the host species oflocust is different. It agrees in the symptoms with which it is associated, its superficial position on the lesions on which it is found, and in its mycelial and conidial characteristics. To this extent it also fully agrees with Giard's (1891 a) description of the Cladosporium-form of his Lachnidium acridiorum. However, nothing has been seen of the Fusarium-form which Giard described. It is difficult to escape the conclusion that Giard's material, dispatched from Algeria to France, showed a secondary growth of a true Fusarium species, and that this or a similar Fusarium persistently contaminated his cultures. Trabut (1891 b) accepted Giard's interpretation of the relationship between the two 'forms' which he saw in his cultures, but nevertheless noted that he never encountered the Fusarium-form on live locusts. Though Brongniart and Delacroix (Brongniart, 1891 d) believed that Giard had probably united several different moulds under one name, they too thought that the Fusarium-form represented the eventual developmental state of the fungus first described by Trabut as B. acridiorum.
The taxonomic position
of the locust fungus
Trabut's (1891 a) disposition of his newly described species in the genus Botrytis is unsatisfactory. Lachnidium acridiorum Giard, which was explicitly established as a new genus and new species, was described from Kunckel & Langlois' material. It was not published as a transfer of Trabut's species even though Saccardo (1892) lists it as L. acridiorum (Trabut) Giard. L. acridiorum was probably based on imperfectly understood material comprising two distinct species of which one was undoubtedly B. acridiorum Trabut. L. acridiorum appears therefore to be a nomen confusum. Fusarium acridiorum (Trabut) Brongniart & Delacroix was proposed (Brongniart, 1891 d) in the belief that a Fusarium state was a stage in the fungus originally described by Trabut. In fact no Fusarium state was initially described by Trabut (1891 a) for B. acridiorum, and the new combination should have been cited as F. acridiorum (Giard) Brongniart & Delacroix being explicitly a transfer of Giard's fungus. The name F. acridiorum should also be disregarded, being based on a nomen confusum. The form and origin of the conidia of the locust fungus here studied and considered to be identical with Botrytis acridiorum Trabut are essentially the same as in Trichothecium roseum (Ingold, 1956), the type species of its genus, except that the smaller conidia of the locust parasite are not regularly produced to the right and left of the apex of the conidiophore. A search of the literature has failed to reveal a description of any species of Trichothecium or allied taxon with which the locust fungus might be synonymous. It is therefore proposed that Trabut's species be transferred to the genus Trichothecium Link ex Fries.
Transactions British Mycological Society Trichothecium acridiorum (Trabut) comb.nov.
Basionym: Botrytis acridiorum Trabut in C. r. hebd. Seanc. Acad. Sci., Paris, lIZ, 1384, 1891. A homonym of B. acridiorum may be a source of confusion. Vuillemin (191 I) referred to Botrytis acridiorum Brongniart & Delacroix, non Trabut. I have been unable to locate the origin of this name, but Vuillemin gives it as a synonym of B. delacroixii Saccardo. This is Saccardo's (1892) name for what Brongniart (189I d) referred to merely as a round-spored form of Botrytis. It is listed by MacLeod (1954) as a synonym of Beauveria bassiana (Balsamo) Vuillemin.
Comparison ofT. acridiorum with Didymopsis locustanae Prinsloo Prinsloo (1962) described a fungus which in South Africa attacked chiefly the intersegmental tissues of the brown locust, Locustana pardalina (Walker). She named it Didymopsis locustanae. Her description of this organism closely resembles the above account of T. acridiorum in respect of the appearance of the colony, and size and general form of the conidiophores and conidia, and the fact that both parasites are weak and do not penetrate the cuticle. The principal point of difference lies in the description of the aerogenous conidia of D. locustanae as being solitary while those of T. acridiorum are in basipetal chains. The early stages of sporogenesis illustrated by Prinsloo resemble comparable stages for T. acridiorum. It is not impossible that D.locustanae and T. acridiorum are the same fungus, for the fragile chains of spores of the latter are readily observed only in slide cultures. A developmental study of D. locustanae would clarify the relationship ofthese species.
Cultural characteristics of Trichothecium acridiorum Though isolates of T. acridiorum grew readily on a wide variety of nutrient media, sporulation was generally lacking or poor on media in common use in mycological laboratories. A systematic test of six isolates on a range of media revealed that except for one strain which remained sterile, all sporulated on BNA and also, to a generally lesser extent, on Sabouraud's Maltose Agar. Both of these media contain peptone. The restriction of the parasite to the surface of locust integument suggested the possibility that it was nourished by materials in the epicuticle and was unable to digest the procuticle. The epicuticle contains lipid material. An experiment was conducted in which the wax from six locusts (N. septemfasciata) was removed by running 1-2 ml. of ether over each and collecting the washings together. The solution oflipids was passed through a Whatman no. I filter-paper to remove suspended particles, and the faintly straw-coloured solution was then spotted repeatedly on to each of a series ofseven clean, polished microscope slides. Evaporation of the solvent left a bloom of wax. The slides were sterilized by storage overnight in propylene oxide vapour. The wax films were then each inoculated with a loopful of suspension of spores from an isolate of the locust parasite. When the droplets had dried the slides were incubated in a saturated atmosphere
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at 23° C. for 3 days, when mycelial growth could be seen at the site of the inoculum. The slides were then transferred to a series of vesselsin which the atmospheric humidity was controlled by saturated salt solution at 80, 84, 87, 88, 92, 97 and 100 % relative humidity ( R .H .) . After incubation for a further 26 days the slides were examined. At 100 % R.H. sparse mycelium had spread 8'5 mm. beyond the edge of the inoculum area and formed an abundance of conidia which were chiefly bicellular, though some were unicellular. Some older parts of the colony had fragmented into arthrospores. At 97 % R .H. the mycelium had advanced 4 mm. but though it had fragm ented into arthrospores it had formed no conidia. At 92 % R.H. and less the fungus had not spread beyond the original inoculum. It is evident that the ether-soluble materials from the locust epicuticleare able to sustain the fungus for vegetative growth and conidium production, and that the fungus though an ectoparasite is not especially tolerant of dry conditions. DISCUSSION
The relationship between Trichothecium acridiorum and the symptoms with which it is associated Though there seems little doubt that the dark lesions on the locusts studied were caused largely or even wholly by T. acridiorum, it has not been possible to prove this. Attempts to infect locusts artificially have not been conclusive because the fungus was apparently almost ubiquitous as a natural contaminant of the locust integument with the result that it always appeared in control populations if environmental conditions were favourable. Attempts to rid locusts of contamination failed. The addition of more spores to the body led to no perceptible increase in incidence of the lesions. The principal evidence for the role of the fungus as cause of the lesions is its constant association, but the possibility remains that the parasite secondarily invades parts of the integument which have already suffered harmperhaps slight physical injury-from some other agency. The parasite tends to become established most commonly on articulating surfaces, e.g. between abdominal sc1erites; but next to this develops most on surfaces which touch the locust's support, e.g. the ventral side of the abdomen and the tarsi. It can occur elsewhere, especially when conditions are humid. It is not clear whether this distribution is the result of mechanical abrasion assisting the establishment of the parasite, or the occurrence of regions of higher humidity, or exposure to sources of infection in the substrate. The following observations are relevant. Containers in which locusts are reared become smeared internally with a film of wax from the bodies of the locusts themselves. It was suspected that in the laboratory the fungus might persist in an active or quiescent state in this film. A clean microscope slide was therefore attached to the inside of such a container and left for 3 weeks. In this period it became smeared generally with a waxy material. It was removed and examined microscopically. Fungal spores resembling the locust fungus were present. The slide was then broken in two and the halves separately placed face down upon malt-agar plates for 3 days at 25°, in which period mould and yeast colonies developed under and around them. Subcultures were prepared
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and several pure cultures of T. acridiorum were obtained. Another slide left for 2 months in a cage containing locusts similarly showed spores morphologically like T. acridiorum in the covering wax film. Except for one spore with a short germ-tube, none had germinated. These observations indicate that though T. acridiorum survived in the wax film which came to line the rearing chamber, it did not actively grow there under the usual environmental conditions. The relationship of the fungus to mechanical injury is not clear, for experiments to investigate this were inconclusive. Locusts are apt to inflict injuries on one another or even on themselves, principally with the tibial spines of the hind limbs, particularly if rather crowded. Abrasion or even puncture by such spines might assist in establishing the fungus, particularly in the lateral folds between the abdominal sclerites. The latter are also sites where the microclimate by being more humid is likely to favour the fungus's growth. It is not at present possible to express an opinion on the relative importance ofinjury and humidity. Apart from wounding, the limbs might also serve as means of transferring spores from the substrate, or from colonies growing among the tarsal pads, to the sides of the abdomen. Although infected locusts died in the laboratory, their death generally followed late upon the appearance of fungoid lesions, and came after a period of diminishing vigour. Owing to the above-mentioned difficulties encountered in conducting infection tests, it has not been possible to decide whether the fungus was the cause of the weakened condition or whether it developed best on locusts ailing from some other cause. It was nevertheless clear that the strictly superficial parasite never caused acute fatal disease. This finding agrees with those of Kunckel & Langlois (189 I ), Trabut (1891 b) and Giard (1892), who refer to the diseased condition as benign. The fungus was found only on adult locusts. No infected nymphs were seen. Possibly the duration of the nymphal instars is too brief to allow the fungus to become established before it is shed with the larval skin at the next moult.
lrifection cycles It is possible that the fungus persists between parasitic phases on laboratory stocks oflocusts in the wax which smears the insides of cages. Rigorous cleaning of the insides of cages with detergent between their use for successive populations oflocusts appeared to reduce the incidence of the disease, but no quantitative data were collected. In so far as the fungus developed well on locusts held under abnormally humid conditions resulting from inadequate ventilation, it seems advisable to aerate laboratory culture chambers adequately if infection is to be minimized. Giard (1892) tentatively ascribed a role in the natural life-cycle of the parasite to the chlamydospores which he saw in his cultures of the Fusarium form of his organism. There is doubt as to which fungus the chlamydospores he saw really corresponded, but the fact that T. acridiorum here formed chlamydospores in culture allows that Giard's suggestion may be correct. In view of the doubt about Giard's Fusarium-form it is unlikely that the genuine Fusarium species reported on eggs of locusts by Marchionatto
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(1933) and Akbar et al. (1958) could have been T. acridiorum. There is still no proof that this parasite of adult locusts also attacks locust eggs. Significance if Trichothecium acridiorumfor biological control Of the workers who were concerned with the fungus in 18g1 when first it was notic ed, Brongniart considered it held high promise as a biological control agent. On th e other hand Kunckel & Langlois (1891) said that it appeared impossible to base hopes of destroying the locusts on the artificial development of this fungus. Trabut (18g1 b) expressed the view that it was insufficiently harmful to be really useful in control. Giard (1892) had similar reservations based on its mild nature, the difficulty in its transmission, and its attack in the field chiefly on spent locusts. The pot entiality of the fungus for biological control of locusts was the subject of a lively public controversy in 18g1 and early 18g2 between Brongniart and Giard and in which the popular press also participated (see Giard, 18g2, for references). The fact that more fungi th an T. acridiorum may have been involved in Algeria (see Brongniart, 1 8g 1 d) may explain some of the conflicting views. There are nevertheless no convincing reports of successful control oflocusts with T. acridiorum. It is reasonable to assume from a review of the relevant literature and from the experience of this present investigation that T. acridiorum holds little pot ential for artificial biological control. I am grateful to Dr M. Norris and other workers at th e Anti-Locust Research Centre, London, for supplying me with infected and healthy locusts. REFERENCES
AKBAR, K. , HAQ.UE, H. & AnBAS, H . M. (1958). Fusarium acridiorum, a parasite of desert locust. Pl. Prot. Bull. FAD, 6, 59. BRONGNIART, C. (1891a ). Les criquets en Algerie. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 131 8- 13 20 . BRONGNIART, C. (189 I b). Telegraph message, dated 19June 1891. C.r. hebd. Seanc. Acad. sa., Paris, 112, 1478. BRONGNIART, C. (189Ic). Le cryptogame des criquets pelerins. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 1494-1496. BRONGNIART, C. (189 1d). Les champignons parasites observes sur les criquets pelerins en Algerie (Acridium peregrinum Oliv .). Bull. Seanc. Soc. natn. Agric. Fr. 51, 627-636. GIARD, A. (1889). Sur quelques typ es remarquables de champignons entomophytes. Bull. scient. Fr. Belg. 20, 197-224. GIARD, A. (189 I a). Sur les Cladospori ees entomophytes, nouveau groupe de champignons parasites des insectes. C.r. hebd. Seanc. Acad. Sci., Paris, 112, 1518-152 I. GIARD, A. (189 I b). Sur Ie champignon parasite des criquets pelerins (Lachnidium acridiorum Gd.). C.r. hebd. Seanc. Acad. Sci., Paris, 113,813-816. GIARD, A. (1892). Nouvelles etudes sur Ie Lachnidium acridiorum, Gd. champignon parasite du criquet pelerin, Revue gen. Bot. 4, 449-461. INGOLD, C. T. (1956) . The conidial apparatus of Trichothecium roseum. Trans. Br. mycol. Soc. 39, 460-464. KUNCKEL D'HERCULAIS, J. & LANGLOIS, C. (1891). Les champignons parasites des Acridiens. C.r. hebd. Sianc. Acad. Sci., Paris, 112, 1465-1468. MAcLEOD, D. M. (1954). Investigations in the genera Beauveria Yuill. and Tritirachium Limber. Can. ]. Bot. 32, 8 18-890. MADELIN, M. F. (1963). Diseases caused by hyphomycetous fungi. Insect pathology, an advanced treatise, vol. II, pp. 233-2 71. Ed. E. A. Steinhaus. New York and London: Academic Pr ess.
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Aires, 34, 227-246. PRINSLOO, H. E. (I g62). Didymopsis locustanae, a new species of fungus on the cuticle of the brown locust. S. Afr. ]. agric. Sci. 5, 23-26. SACCARDO, P. A. (r892). Svlloge fungorum, IO, 964 pp. TRABUT, L. (18gla). Sur une maladie cryptogamique du criquet pelerin (Acridium pere-
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VUILLEMIN, P. (1911). Les Isaria de la famille des Verticilliacees (Spicaria et Gibellula).
Bull. Soc. mycol. Fr. 27, 75-82. WOLLENWEBER, H. W. & REINKING, O. A. (1935). Die Fusarien, ihre Beschreibung, Schadwirkung und Bekdmpfung, 355 pp. Berlin: Paul Parey.
(Accepted for publication
20
July 1965)