A parasite of white cysts of Heterodera: Catenaria auxiliaris

Trans. Br, mycol. Soc. 69 (3) 367-376 (1977)

Printed in Great Britain

A PARASITE OF WHITE CYSTS OF HETERODERA: CATENARIA AUXILIARIS By H. T. TRIBE Department of Applied Biology, University of Cambridge Tarichium auxiliare KUhn was described from females of the beet cyst-nematode Heterodera schachtii Schmidt as a mycelial fungus and resting spores. The resting spores are shown to belong to a rhizomycelial fungus which forms them singly inside resting sporangia and also forms posteriorly uniflagellate zoospores inside zoosporangia. The resting spore part of Kuhn's fungus is transferred to the genus Catenaria, but his mycelial fungus can not be characterized. Aspects of the nature, biology, distribution and relationships of C. auxiliaris are discussed. The fungus is widespread in populations of H. schachtii and is also known in the cereal cyst-nematode Heterodera avenae.

The sugar beet cyst-nematode, Heterodera schachtii Schmidt, was a devastating parasite in the early years of the European sugar industry before it was brought under control by crop rotation. Roots of beet are invaded by the vermiform larvae which pass completely inside, become sedentary, feed and grow, the females swelling to the extent of bursting out of the root. Mature females are visible to the naked eye on the roots as nodules or 'white cysts', 0'3-1'0 mm diam, and die as they transform into brown cysts, which consist of the content of eggs enveloped in the hardened, darkened cuticle. The cysts fall off the root and persist in the soil as propagules, from each of which several hundred larvae later emerge to recommence the life-cycle. KUhn (1877) gave a preliminary report on the causes of beet-sickness of soil and investigations of the nature of nematodes. Included as a brief part of the report was a paragraph on what he believed was a single fungus parasitic upon the female nematode and after noting that this nematode was not attacked by any animal parasite he described it thus (translated from the German): It is therefore the more significant that in our investigations plant parasites were found, not uncommonly, in the female nematodes, appearing in two different forms. The most frequent form is a mycelial fungus, whose water-clear hyphae bear small uncoloured conidia and richly invest the eggs in the interior of the female. Often all the eggs are disorganized by the parasite; not rarely the embryos succeed in developing, but are destroyed in the eggs, and only individuals escape the corruption. Frequently the fungal hyphae project from the hind part of the females through the anus. The second parasitic form is characterized by great yellow-brown spores, which

have an areolate outer skin and look to some extent similar to the spores of the couch grass smut, Tilletia controversa mihi. Where they appear the eggs and embryos are wholly destroyed and the bodies of the females are stuffed full with them. As far as I could elucidate the developmental process, I have good reason to regard both forms as probably belonging to the same species. The latter form represents resting spores which mediate reproduction from one year to another, whilst the mycelial fungus is the conidial form which serves multiplication during vegetative growth. It is probable that this parasite forms the type of a genus of its own; as a preliminary I have placed it into that genus which stands nearest - it may therefore bear the name Tarychium auxiliarum. Kuhn (1881) published a full report on the researches but added nothing concerning the parasitic fungus: on the contrary he merely gave a shortened form of the preliminary account and corrected the spelling of the name to Tarichium auxiliare. The resting spores were next found in Russia by Korab (1929) who examined brown cysts sieved from soil and not females on roots. The fungus only inhabited empty cysts, and he believed it grew in mucilage present inside them. He therefore considered it unimportant as a parasite despite its fairly frequent occurrence and the presence of large numbers of resting spores inside cysts. Korab was the first to illustrate the spores, but his photomicrograph is at insufficient magnification to show detail of the markings. The first British record was made by Jones (1945). Incidental to examining a great many brown cysts of Heterodera schachtii from fen soils of East Anglia, he noticed four different symptoms

Catenaria auxiliaris on Heterodera cysts of disease which he presumed were brought about by fungal invasions. One of these was represented by a single cyst, which was devoid of eggs and larvae, but contained more than 100 black, strongly sculptured spores similar in appearance to those of bunt of wheat (Tilletia caries). Some of the spores were spherical, others ovoid, the average sizes being: spherical, 36/lm, ovoid, 45 x 23 /lm. In a treatment of insect pathogenic fungi originally described from their resting spores mostly as Tarichium species, MacLeod & MullerKogler (1970) discussed T. auxiliare as known from Kuhn's description but excluded it from Entomophthora (Tarichium) as an insufficiently known species. Bursnall & Tribe (1974) reported finding large reticulate yellowish or brownish spores similar to the description of T. auxiliare in MacLeod & Muller-Kogler (1970). The spores were found in only two of 800 brown cysts and were noted to be mostly larger than the given dimensions. MacLeod & Muller-Kogler have assumed the resting spores of T. auxiliare were the same size as the spores of Tilletia controversa KUhn, which they cited as 17-22 /lm, but Kuhn did not specify a size and simply described the spores as somewhat similar (' einigermassen ahnlich ') to those of his smut fungus. The size of the resting spores KUhn saw is therefore not known. Recent studies have shown that the resting spore fungus of KUhn is widespread in populations of Heterodera schachtii and more is now known of its life-history. There is no connexion with Kuhn's mycelial fungus. DESCRIPTION

The fungus is parasitic in the female nematode (= white cyst) but will not enter the membranes of eggs whether inside the growing female nematode or inside the matured and dead body (= brown cyst). The process of infection has not been observed. The first stage so far seen is young rhizomycelium characteristic of Catenaria inside the female nematode. Illustrations of this and other main features of the species are given in Figs. 1-14. In unstained mounts in absence of phase contrast the rhizomycelium is difficult to see amongst the richly globular host cytoplasm. The apical cell of a length of rhizomycelium is generally narrowed especially at the tip but the next and subsequent cells are swollen. They bear fine rhizoids and are separated by clear septa. Unswollen lengths of hypha between the swellings (isthmuses) are absent, although the shape of some rhizomycelial swellings is narrow near the septa. At maturity each swollen segment becomes a precursor

sporangium which converts either into a zoosporangium or a resting sporangium. The rhizoids are lost and the segment rounds off so completely that nothing of the previous connections with neighbouring hyphal swellings is visible on the precursor sporangium. Precursor sporangia are usually spherical or subglobose but may also be ovoid or somewhat elongated. The dimensions are variable within the range 20-50 um, generally between 25 and 45/lm, with means of individual populations sometimes substantially above or below 35/lm. Zoosporangia have been seen in only 2 of 14 populations of the fungus found infecting white and young brown cysts on rootlets and in none of 16 populations found in brown cysts sieved from soil samples (Table 1), although precursor sporangia have been sometimes found in the latter. As the cytoplasm divides into zoospores, several papillae, typically about six, form on the sporangial surface. They soon gelatinize and dissolve, allowing the zoospores to emerge, after which event the escape pores are clearly visible. The zoospore is posteriorly uniflagellate, and globose, ovoid or elongated, approximating 3 /lm, 3'5 x 2'0-2'5 /lm and 5 x 2 /lm respectively, with the flagellum 12-15 /lm long. The zoospore swims for a short distance in a straight line and then suddenly changes direction, sometimes dithering for a moment at the change. Several globules, bright white under phase contrast, may be visible at the anterior end of the spore, but are not always evident. Resting sporangia were present in all 30 populations examined. A single reticulate spore is formed inside each sporangium, in some instances contracting markedly so that it is free within the sporangial wall (aplerotic) but in others not so contracting (plerotic). The resting spore takes its shape and size from the sporangium, It is thus typically globose to subglobose but may be ovoid, oblong, pyriform or reniform, and of the same size, as, or rather smaller than, the sporangium. Germination has not been seen, although after placing spores in water the reticulate layer has in a few instances cracked to reveal an inner membrane. The resting spore shows a striking superficial resemblance to the chlamydospore of Tilletia contraversa Kuhn (cf, photomicrographs of the latter by Conners, 1954), and the present author has no hesitation in identifying the nematode parasite with the resting spore fungus described by KUhn. The mesh patterns (areolae) on the smut spore are similar to those of the nematode fungus, but the smut spore is smaller, measuring 16-19/lm according to Kuhn's diagnosis (quoted

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Table 1. Records of Catenaria auxiliaris showing incidence in populations of the host cyst-nematode, Heterodera schachtii Location and date examined rr:

England 1974

England 1975

Sweden 1975

England 1973-4

Europe 1975 England Netherlands Germany

Czechoslovakia

-,

----'--

No. cysts examined

No. cysts parasitized

In white and brown cysts taken from rootlets. Active phase of parasite Oxlode Cambridgeshire July 56 3 18 Cambridgeshire July Reach 1 158 1 Cambridgeshire Reach 2 July 63 10 2 Swaffham Prior 1 Cambridgeshire July 1 Aug. Littleport 1 Cambridgeshire 45 Aug, 2 Littleport 2 Cambridgeshire 30 Sept, 204 Cambridgeshire Reach 1 4 Sept, 1 Reach 2 Cambridgeshire 99 Sept, 2 Lakenheath 1 Suffolk 27 Epworth 1 Lakenheath 2 Lakenheath 1 Epworth 1 Alnarp

Sth Humberside Suffolk Suffolk Sth Humberside near Malmo

July July July Oct. Sept.

422 35 83 145 139 1516

51 2 2 21 1 111

Percentage infection 5'4 11'4 1,6 20'0 2'2 6'7 2'0 1'0 7'4 12'1 5'7 2'4 14'5 0'7 7'3 2

In brown cysts sieved from soil samples, Passive phase of parasite Suffolk Nov. Lakenheath 3 4 530 1 Swaffham Prior 2 Cambridgeshire Jan. 46 1 May Yaxley Cambridgeshire 15

0,8 2'2 6'7

Southery Feltwell Steenbergen Dinteloord Neuss 1 Elsdorf 1 Elsdorf 2 Elsdorf 3 Semcice

Norfolk Norfolk Noord Brabant Noord Brabant Rhineland Rhineland Rhineland Rhineland near Mlada Boleslav

Jan, Jan, Apr. Apr, Mar. May Dec. Dec,

310 183 135 75 241 100 112 100

1 1 6 1 8 1 1 11

0'3 0'5 4'4 1'3 3'3 1'0 0'9 11'0

June

80

1

1'3 10'2 3'0

U.S,A,1975

Chualar Spreckels

California California

July July

127 67

13 2

Germany 1976

Neuss 2 Neuss 3

Rhineland Rhineland

Apr. Apr,

100 106 2327

1 1 54

in Conners, 1954), 14'5-19'5 JIm in the several specimens examined by Conners and 17-22 p.m according to MacLeod & Muller-Kegler (1970). Considering in conjunction the catenate rhizomycelium and the zoosporangia with posteriorly uniflagellate zoospores, the nematode parasite is placed, at least provisionally, as a new combination in Catenaria Sorokine. Now that the resting spores are known to develop in rhizomycelium it is certain that the mycelial fungus described by KUhn and believed by him to be part of the resting spore fungus was unconnected with Catenaria auxiliaris.

1-0 0'9 2'32

Kuhn's epithet derives from his view that control of beet cyst-nematode was brought about primarily by crop rotation and control of weeds susceptible to the nematode, with the fungus behaving as valuable auxiliary troops which assisted in the destruction of the lowered populations of nematodes surviving the main control: ' ... die Nemato den auch ihre nanirlichen Feinde haben, die sich unseren Vertilgungsbestrebungen als werthvolle Hilfstruppen erweisen werden' (Kuhn, 1881).

37°

Catenaria auxiliaris on Heterodera cy sts

Catenaria auxiliaris (Kuhn) comb.nov, Tarychium auxiliarium Kiihn pro parte, Z. Ver. Rubenz .-Industr. Dtsch Reich 457 (1877) . Tarichium auxiliare KUhn pro parte, Ber. physiol. Lab . Versuch. lands», Inst, Univ. Halle 3: 136 (1881). Parasitic in female nematodes of Heterodera schachtii Schmidt and H. avenae Wollenweber. A catenate rhizomycelium develops inside the nematode, consisting of swollen portions delimited by septa and without isthmuses. The swellings develop into precursor sporangia of globose, subglobose or less often oblong, ovoid or similar approximately spheroidal shapes. The precursor sporangia measure mostly 25-45 flm diam and mature into either zoosporangia or resting sporangia. Zoosporangia are less commonly found. They dehisce by forming about six gelatinous papillae through which the posteriorly uniflagellate zoospores escape. The zoospore body is plastic and varies in shape from globose or ovoid to elongate and in size from approximately 3 flm diam to 3'5 x 2'5 to 5 x 2 flm respectively. The flagellum is 12-15 flm in length. Resting sporangia are constantly found and each develops internally a single plerotic or aplerotic resting spore, whose wall is 1'5-3 flm thick and made up of mesh-like areolae separated by ridges giving a reticulate appearance. The spore thus resembles the chlamydospores of the smuts Tilletia controuersa Kuhn and T . caries (DC.) Tu!. The resting spore is yellow-brown, or paler or darker, shaped as its precursor sporangium and of equal or slightly smaller size. Germination has not been seen. Slides of Catenaria aux iliaris have been deposited at the Commonwealth Mycological Institute, Kew (1MI 212206) and the Centraalbureau voor Schimmelcultures, Baarn, Netherlands. They function as neotypes since it is virtually certain that no material was preserved.

BIOLOGY

C. auxiliaris is a parasite of the female cystnematode and observations on naturally occurring material indicate that if the nematode is young at the time of invasion it is entirely destroyed and replaced by sporangia . Those females in which eggs are being formed are invaded and largely destroyed but egg membranes are never penetrated. In older nematodes large numbers of eggs may be present between the Catenaria sporangia, including eggs containing larvae, and it is possible that these can hatch . In females invaded when nearly mature the nematode cuticle turns brown and converts into a cyst containing viable nematode eggs and Catenaria resting spores. The spores are apparently dormant and unable to invade the eggs, which explains the observations of Korab (1929) that the fungus appeared to be a saprophyte in brown cysts. Nematode larvae in such cysts hatch normally, leaving old cysts empty except for egg shells and reticulate resting spores. C. auxiliaris grows parasitically upon the tissues of the cyst-nematode, which, in old females is degenerating into mucilage. Young female nematodes taken from the host rootlet, placed in a hanging drop of water in a van Tieghem cell, and inoculated with zoospores from vigorously dehiscing zoosporangia, have been infected with C. auxiliaris. Figs. 4 and 5 were prepared from a cyst-nematode so infected after 3 days at summer laboratory temperatures . Subsequentlya group of 21 females, collected from root systems on which all other cysts examined were healthy, and which were themselves presumably healthy, were placed, three each, in 7 hanging drops and inoculated with zoospores. A female was taken from each drop and crushed to examine for rhizomycelium after 2, 3 and 4 days. None of seven revealed rhizomycelium after 2 days, three of seven did so after 3 days and five of seven after

EXPLANATION OF FIGURES

Carenaria auxiliaris All stained preparations have been mounted in 0'05 % trypan blue in lactophenol. Fig. 1. Apical portion of rhizomycelium showing swellings bearing rhizoids. Note oil released from the infected nematodeduring preparation of the slide. Stained. x 550. Fig. 2 . Older rhizomycelium from the samepreparation showing lossof fine rhizoidsand septa delimiting swellings, in rich oily nematode cytoplasm. Stained. x 550. Fig. 3. Young rhizomycelium dissected from very young infected female showing rhizoids and septa. Note ready detachmentof the segments. Stained. x 550. Fig. 4. Rhizomycelium in female 3 days after infection by zoospores in a hanging drop of water. Unstained in water. x 1050. Fig. 5. Maturing segments of rhizomycelium without rhizoids,3 days after infection with zoospores in a hangingdrop. Stained. x 1050. Fig. 6. Precursor sporangia in cyst. Unstained in water. x 550.

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Catenaria auxiliaris on Heterodera cysts

372

12

H. T. Tribe 4 days. It is not possible to detect early infection of a female without squashing to reveal the rhizomycelium. No growth has so far been obtained on agar. Drops of water containing zoosporangia and zoospores were placed on to the surface of cornmeal agar and the plates tilted so that the drops ran across the surface. No sign of growth was seen on subsequent observation under the microscope. This failure was unexpected, for if the fungus is able to grow in mucilage resulting from degenerating nematode tissues it should be capable of growth on artificial media. Lack of further zoosporic material prevented further tests. Resting spores have never been induced to germinate. Three successive trials were conducted in which groups of resting sporangia were placed in contact with female nematodes attached to portions of host rootlet and laid on to moist soil. All failed. A later trial failed when groups of spores were placed in contact with female nematodes on the living host plant. Resting spores failed to germinate on plates of cornmeal agar even after intermittent illumination under a light bench or treatment with periods of refrigeration at 2 DC. They failed to germinate in hanging drops of water even when pre-stored for up to 6 months at 2 or after heat shocks at 80 The role of the resting spore is thus obscure. The rarity of the zoosporic state is also noteworthy. Suspecting that zoospores might be produced by this fungus the author had several times placed precursor sporangia in hanging drops of water in 1974, but without positive result. In the summer of 1975 two of the three samples available yielded zoospores, the sample from Epworth (T able 1) in very large numbers. Another sample sent from the same site in October 1975 however yielded no zoosporangia despite the presence of plenty of rhizomycelium and pre0

0

,

•

373

cursor sporangia. Unfortunately no slide preparations of zoospores were made at the time of abundance and data concerning the zoosporic state is based on observation of much less material than that on resting sporangia. It is possible that the unusually dry summers of 1975 and 1976 have hindered production of the zoosporic state and indeed reduced the incidence of the fungus on populations of its host. C. auxiliaris has been found in females of Heterodera avenae Wollenweber (Kerry, 1974, 1975) but has not yet been reported in other species of Heterodera. There is no evidence as to whether the vermiform male nematode of Heterodera is invaded . Eggs of H. schachtii are not invaded by rhizomyceliurn, but it is conceivable they could be penetrated by zoospores. This could explain the resting spores illustrated inside an egg by Bursnall & Tribe (1974, Fig. 5). Circumstantial evidence of infection of free-living vermiform nematodes is negative. Fungal parasitism of these nematodes has been much studied and failure to have observed the highly characteristic resting spores by investigators is most unlikely. The fact that a century has passed between this account and KUhn's first report of this fungus is largely due to the circumstance that those interested in pathology of Heterodera have studied mature cysts from soil samples rather than developing females and cysts upon the host roots. Through examination of females, Kerry (1974) has discovered a fungus with entomophthoraceous affinities in H. avenae and the author has found a similar fungus in a single sample of H. schachtii from Sweden (Tribe, 1977 a). No entomophthoraceous fungi have earlier been described in H. schachtii, although the fungus illustrated by Rozsypal (1934) as ' Olpidium nematodae' may well have been such (T ribe, 1977b).

EXPLANATION OF FIGURES

Cat enaria auxiliaris

All stained preparations have been mounted in 0'05 % trypan blue in lactophenol. Fig. 7. Zoosporangium in water showingsuccessive stages of dehiscence. Unstained in water. x 550. Fig. 8. Empty zoosporangium showingexit pores. Unstained in water. x 550. Fig. 9. Precursor sporangia, two empty zoosporangia and a resting sporangium with aplerotic spore. Unstained in lactophenol. x 550. Fig. 10. Young resting sporangia and mature sporangia with aplerotic spores. Stained. x 300. Fig. 11. Mature resting sporangia with plerotic spores. Stained. x 300. Fig. 12. Mature resting sporangiumwith apleroticspore in median focus. Stained. x 1050. Fig. 13 . Mature resting sporangiawith plerotic spores in median focus. Stained. x 1050. Fig. 14. Resting spore, with reticulate wall split and revealing inner membrane. Unstained in water. x 550.

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Catenaria auxiliaris on Heterodera cysts DISTRIBUTION

C. auxiliaris is widespread in populations of Heterodera schachtii. Of 29 samples of white and young brown cysts examined on rootlets of infested sugar beet over a 3-year period, 14 revealed C. auxiliaris. Of mature brown cysts sieved from a total of 114 soil samples, 16 populations showed the presence of C. auxiliaris. The locations of the positive samples with their incidence of infection are given in Table 1. The overall average level of infection of young cysts taken from rootlets was 7'3 %, appreciably higher than the level of 2'4 % in cysts sieved from soil. This is expected, because only C. auxiliaris infections of mature females will be detectable in mature cysts sieved from soil, for only such females will succeed in forming persistent cysts. The fact that a population of sieved cysts contains C. auxiliaris indicates that many young nematodes were destroyed earlier on the host rootlets. The numbers following certain locations cited in Table 1 show that the samples came from the same source and often from the same site. C. auxiliaris was unexpectedly rare in certain experimental fields at Elsdorf, Germany, which were sampled after 11-12 years monoculture in sugar beet. This cropping should have encouraged its incidence if present, and the finding of one cyst containing resting spores indicated, though minimally, that C. auxiliaris was present. The single infected cyst was found in one of six populations of mature cysts examined in 1975 (total 673 cysts); it is represented in Table 1 as 'Elsdorf 1'. 'Elsdorf 2 and 3' represents two of three nonmonoculture samples examined from different sites in Elsdorf. No young cysts in four monoculture and two non-monoculture populations (total 360 cysts) from Elsdorf examined in 1976 were infected with C. auxiliaris (Tribe, 1978). C. auxiliaris was not found in small numbers of cysts of Heterodera aoenae (cereal cyst-nematode), H. trifolii (clover cyst-nematode) or H. glycines (soya bean cyst-nematode) examined by the author (Tribe, 1977a), but Kerry (1975) found it in H. avenae females from 4 of 21 soils examined from southern England. RELATIONSHIPS

The fungus to which C. auxiliaris is most nearly related is undoubtedly Catenaria spinosa Martin. C. spinosa is a parasite of eggs of an unidentified species of midge belonging to the genus Chironomus. The host egg masses were collected from a stream in Virginia, U.S.A., in 1973 (Martin, 1975)·

In both these species the matured rhizomycelial segment converts either into a zoosporangium or into a resting (= resistant) sporangium. For the matured segment in C. auxiliaris the writer has used the term 'precursor sporangium', because it is a distinct morphological entity (Fig. 6) which will form one or other type of sporangium. Which type is to be formed can not yet be predicted in advance. If a resting sporangium is formed, this may either develop a reticulate resting spore of smaller size than the sporangium and thus easily visible inside the sporangial wall, or mature without shrinkage of contents to form a spore morphologically identical with the sporangium. In the former case, there is a clear distinction between resting sporangium and resting spore. The writer does not accept the terminology used fairly generally by students of lower phycomycetes and by Martin (1975) in his discussion relative to the genus Catenaria,where the term' container' is used for resting sporangium and 'resistant (or resting) sporangium' for resting spore. The resting sporangium is clearly alternative to and analogous with the zoosporangium, and the resting spore analogous with the content of zoospores. Where there is no evident distinction between resting sporangium and resting spore, the writer prefers to view the matured structure as a resting sporangium containing a plerotic resting spore. Resting sporangia of C. spinosa differ markedly from those of C. auxiliaris in being covered with spine-like processes apparently derived from rhizoid bases. The sporangia are not reticulate, they do not mature discrete (aplerotic) spores and they are less common than the zooosporangia, having been induced to form only after extended exposure of the host egg-masses to the relatively low temperature of 15°. Their germination has not been seen. Zoosporangia in C. spinosa are marginally larger than those in C. auxiliaris (2569 x 24-66 pm, mean 43 x 40 pm), mature larger zoospores (7-11 x 4'5-7'5 pm, with flagellum up to 34 pm) and form fewer exit papillae (1-3). Martin (1975)noted that C. spinosa differedfrom C. anguillulae Sorokine, C. allomycis Couch and C. sphaerocarpa Karling in lacking elongated sporangial discharge tubes, and from C. anguillulae and C. allomycis in producing typically rounded zoosporangia with several openings. In both these characters it resembles C. auxiliaris. A feature it shared only with C. sphaerocarpa was the production of plerotic resting spores. C. auxiliaris forms both aplerotic and plerotic resting spores, and examination of more collections of C. spinosa may well show populations in which aplerotic resting spores occur. Martin further noted that C. sphaerocarpa was dismissed from Catenaria

H. T. Tribe by Couch (1945) because the zoospores were chytrid-like, although Sparrow (1960) had retained it within the genus. Concerning the swimming habits of C. sphaerocarpa zoospores, Karling (1938) had written that they 'jerk and dart about a great deal in swimming, come to rest momentarily, and then dart off again in another direction'. This exactly describes the swimming habit of C. auxiliaris zoospores. Martin states that zoospores of C. spinosa swim smoothly in water, making no mention of direction changes, so that in this property the two species differ. More data js needed on the structure of the zoospore in C. auxiliaris. Two species of Catenaria were not noted by Martin. C. uerrucosa Karling isolated from one soil sample on snake skin bait, has only once been seen. It was characterized by verrucose, aplerotic resting spores, but motile zoospores were not observed (Karling, 1966a). C. vermicola Birchfield was isolated from vermiform nematodes (Birchfield, 1960) but from the description seems scarcely separable from C. anguillulae. C. anguillulae, the type species, was described by Sorokine (1876) on nematodes which had developed amongst algae in a vase of water. It is also known from eggs of liver flukes, helminths, mites and rotifers, and from adult rotifers (Sparrow, 1960). It infects embryos and eggs of Daphnia magna but can not infect adult animals before their death (Gaertner, 1962). Good photomicrographs of it in male (vermiform) nematodes of Heterodera rostochiensis are given in an article by Jones in Price-Jones & Solomon (1974, p. 263). I have never seen it in females or matured cysts of any Heterodera. It grows readily as a saprophyte and has been isolated from soils on baits of bleached corn leaves, onion skin, hemp seed, snake skin and pine pollen (Karling, 1966 b). Several quite large differences therefore exist between C. angui/lulae, the type species of Catenaria, and both C. auxiliaris and C. spinosa. The author evaluates the following as the more significant. The zoosporangium of C. anguillulae is not a typically rounded structure and generally forms a single exit tube through which the zoospores usually emerge in a cluster. That of C. auxiliaris and C. spinosa is typically rounded and generally forms several exit papillae through which the zoospores leave individually. The resting spore of C. angui/lulae germinates fairly readily to form an exit tube through which zoospores emerge. Those of C. auxi/iaris and C. spinosa have not yet been germinated. C. angui/lulae grows in very diverse hosts and in agar media, with its morphology varying a great

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deal according to host or substrate. Both other species, whose resting structures have a characteristic ornamentation and should readily be observed should they occur in other organisms, seem limited as to host. Further investigation of C. auxiliaris and C. spinosa is necessary before these differences can be firmly established. Should they be confirmed the two species may better be placed in a new genus, as suggested for' Tarichium auxiliare' by Kiihn just one hundred years ago. The author is greatly indebted to those many individuals who have kindly sent him material of Heterodera schachtii. He thanks Dr E. MiillerKogler of Darmstadt for early discussion of this fungus and for providing photostats of the relevant parts of Kiihn's reports, Dr Brian Cooper of the Commonwealth Bureau of Plant Genetics in Cambridge for sorting out the title of the journal in which Korab's paper was published and translating the relevant parts from the Russian. He acknowledges the assistance of Miss Diana Waterson in examining some samples of H. schachtii for the fungus, and the Perry Foundation for financial support of the study of cyst-nematode pathology, of which this contribution forms a part. REFERENCES

BIRCHFIELD, W. (1960). A new species of Catenaria parasitic on nematodes of sugarcane. Mycopathologica et Mycologica applicata 13, 331-338. BURSNALL, L. A. & TRIBE, H. T. (1974). Fungal parasitism in cysts of Heterodera. II. Egg parasites of H. schachtii. Transactions of the British Mycological Society 62, 595-601. CONNERS, I. L. (1954). The organism causing dwarf bunt of wheat. Canadian}ournal of Botany 32, 426431. COUCH, J. N. (1945). Observations on the genus Catenaria. Mycologia 37, 163-193. GAERTNER, A. (1962). Catenaria anguillulae Sorokine als Parasit in den Embryonen von Daphnia magna Strauss nebst Beobachtungen zur Entwicklung, zur Morphologie und zum Substratverhalten des Pilzes. Archiv fur Mikrobiologie 43, 280-289. JONES, F. G. W. (1945). Soil populations of beet eelworm (Heterodera schachtii Schm.) in relation to cropping. Annals of Applied Biology 32, 351-380. KARLING, J. S. (1938). A further study of Catenaria. American}ournal of Botany 25, 328-335. KARLING, J. S. (1966a). Some zoosporic fungi of New Zealand. X. Blastocladiales. Sydowia 20, 144-150. KARLING, J. S. (1966b). The chytrids of India, with a supplement of other zoosporic fungi. Beiheft zu Sydowia 6, 1-125. KERRY, B. R. (1974). A fungus associated with young females of the cereal cyst-nematode, Heterodera avenae. Nematologica 20, 259-260.

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(Accepted/or publication 9 May 1977)