Alan F. Bird

Observations on the use of insect parasitic nematodes as a means of biological control of root-knot nematodes

Abstract Observations on the use of insect parasitic nematodes as a means of biological control of root knot nematodes. International Journal for Parasitology16: 511–516. Infective larvae of the entomophagous nematode Steinernema glasen have been shown to orient towards the root tips of germinating tomato seedlings on 0.75% aqueous agar. This behaviour parallels that of the infective larvae of the root-knot nematode Meloidogyne javanica under similar conditions in that these larvae also accumulate in the meristematic region of the root. In both instances it seems that carbon dioxide is largely responsible for this orientation. Our pot experiments showed that S. glaseri, when applied at high concentration (5 × 106 per plant) at one application or at a lower application (5 × 105) daily over 10 days, significantly reduced the numbers and reproductive capacity of M. javanica applied at a concentration of 2 × 103 per plant. This effect was approx. 4-fold when expressed as a total egg mass count and approx. 6-fold when expressed as egg masses per gram of fresh wt of root. In addition to reducing the infestation of plants by root-knot nematodes the application of larve of S. glaseri led to the production of significantly larger plants. The nature of the behaviour of larvae of S. glaseri that brings about this reduction in numbers of M. javanica that have developed to the egg-laying stage is discussed.

Quantitative studies on the growth of syncytia induced in plants by root knot nematodes

Abstract Bird A. F. 1972. Quantitative studies on the growth of syncytia induced in plants by root knot nematodes. Internationaljournal for Parasitology2: 157–170. Growth of the root-knot nematode (Meloidogyne javanica) in several different susceptible hosts was measured together with that of the tumour-like syncytia induced by each nematode. Size and DNA content of the syncytial nuclei were also measured. Growth of the nematode was most rapid between the completion of moulting and the commencement of egg laying (2–4 weeks) and continued at a decreased rate for the rest of the experiment (4–7 weeks). Syncytial and nuclear growth and DNA content reached a peak just before egg laying started (2–3 weeks). After this time the size of the syncytia as well as the combined area of their nuclei and the DNA content of the nuclei declined significantly. The DNA content of individual syncytial nuclei was extremely variable at all harvests.

Studies on the surface coat (glycocalyx) of the dauer larva of Anguina agrostis.

Imprints of the surface coat (glycocalyx) from the cuticles of living second stage dauer larvae (DL2) of Anguina agrostis (syn. A. funesta) have been examined using incident light fluorescence microscopy and scanning electron microscopy. These surface coats contain residues of N-acetyl-D-glucosamine which were detected by treatment with wheat germ agglutinin labelled with either fluorescein or rhodamine. They also contain protein which was demonstrated by treatment with either pepsin or trypsin. These enzymes inhibited the attachment of the coryneform bacterium Clavibacter sp. to the surface coat, indicating that proteins play a crucial role in the adhesion of these bacteria to the nematode. This inhibition of attachment was reversed within 18 h after removal of the DL2 from the enzymes, indicating that the nematode was capable of renewing its surface proteins.

Adhesion of conidia of the fungus Dilophospora alopecuri to the cuticle of the nematode Anguina agrostis, the vector in annual ryegrass toxicity.

Abstract The adhesion of conidia of the fungus Dilophospora alopecuri to the surface of the second stage dauer larva (DL2) of the nematode Anguina agrostis (syn. A. funesta) was examined using both light and electron optics. The process of attachment does not lead to any apparent damage to the epicuticle of the nematode. Photographs of sections cut tangentially through the setulose appendages of the conidia show that a mucilagenous fibrillar material appears to be exuded from the highly convoluted surface of these appendages. This material adheres to the surface of the nematode cuticle and is deposited in the transverse annulations. The adhesion of these spores to DL2 of A. agrostis was examined in 4822 nematodes from four galls. The mean percentage of DL2 with spores adhering was 64% and ranged from 43 to 85%. This adhesion was compared with that of Corynebacterium rathayi from bacterial galls and was found to coincide. Thus, bacteria adhere to nematodes with D. alopecuri conidia attached and these conidia adhere to nematodes with C rathayi attached. Furthermore, DL2 that are free from conidial adhesion appear to be free from bacterial adhesion and, in most instances, DL2 that remain from from bacterial adhesion remain free from conidial adhesion. These observations draw attention to the potential of D. alopecuri as an agent for the biological control of annual ryegrass toxicity. Conidial adhesion to A. agrostis differs from bacterial adhesion to this nematode in that no visible damage to the cuticle takes place. The concept that adhesion of microorganisms to nematodes occurs in two phases, one involving site recognition and the other, if it occurs, involving physiological interaction and morphological change is discussed.

Moulting of parasitic nematodes.

Abstract Nematodes are usually divided into two major groups, the Adenophora which are common in water and the Secernentea largely from soil. Most research on moulting has been done with the Secernentea, which include the majority of the parasitic forms. Cuticle composition and morphology is discussed in relation to the physiology and possible reasons for moulting in the Nematoda.

Cuticle printing of nematodes.

Abstract The cuticle surfaces of taxonomically widely divergent species of nematodes have been shown to be covered by a surface coat (glycocalyx) that contains carbohydrate. This surface coat is not considered to be part of the nematode cuticle but to be secreted by the nematode onto its surface. In these studies it is shown that the surface coat of the second stage infective dauer larva (DL2) of the ryegrass nematode, Anguina agrostis (syn. A. funesta), leaves an imprint which is an extremely delicate structure that under normal conditions is not visible under the light microscope. However when treated with the lectin wheat germ agglutinin (WGA) labelled with fluorescein isothiocyanate (FITC) this imprint becomes clearly visible under blue or ultra violet (u.v.) light and resembles a micro finger print.

Plant-parasitic nematodes.

It is becoming increasingly apparent to many researchers that both plantand animal-parasitic nematodes face very similar biological challenges in interacting with their respective hosts. Although the precise nature of the molecules mediating key aspects of the host–parasite interaction will almost certainly be different in the different kingdoms, the underlying principles will be the same, and model systems based on plant-parasitic nematodes offer certain practical advantages over those involving animal hosts in elucidating these principles. In particular, the development of the soybean cyst nematode (SCN) as a genetic model (Dong and Opperman, 1997), in conjunction with the ability to manipulate host plants by forward and reverse genetics, permits these powerful techniques to be employed to dissect the host–parasite interaction. The burgeoning deployment of genomics in studies of parasitic nematode biology (Blaxter, 1998; Opperman and Bird, 1998; Bird et al., 1999) will provide the tools to link SCN genetics to less tractable parasitic species, including animal parasites. Parasite genetics are discussed in Chapters 3, 4 and 5. It is obviously not possible to provide a comprehensive review of plant nematology in one chapter, and readers are directed to descriptions of the taxonomy (Nickle, 1991), morphology (Bird and Bird, 1991), physiology and biochemistry (Perry and Wright, 1998) and cell biology (Fenoll et al., 1997b) of plant-parasitic nematodes. Rather, examples will be provided from plant-parasitic nematodes that emphasize the catholic nature of nematode parasitism. Understanding how the host and parasite communicate,

Physiological and morphological studies on secretion of a protein-carbohydrate complex by a nematode

Abstract Dropkin V. H. and Bird Alan F. 1978. Physiological and morphological studies on secretion of a protein-carbohydrate complex by a nematode. International Journal for Parcsitology 8 : 225–232. Secretion of the gelatinous matrix by females of Meloidogyne javanica was induced by compounds extracted from the roots of either susceptible or resistant plants. The stimulus that induced this secretion was not specific but analysis of root extracts implicated the nucleic acids. DNA was the most active substance tested (minimum concentration = 0·0075 mg/ml). The response was rapid, usually within 10 min and did not appear to be nerve mediated. The shape of the exudate in vitro was conditioned by the composition of the medium in which the nematodes were tested. Stimulation brought about significant changes in the nuclei of the rectal gland cells which enlarged, in the nucleoli, which tended to become more granular and vacuolated, and in the cytoplasm, where there was an increase in the number of Golgi bodies per unit area.

Vital staining of glycoprotein secreted by infective third stage larvae of Haemonchus contortus prior to exsheathment

Staining of infective third stage larvae of Haemonchus contortus prior to exsheathment shows that protein is exuded from the head region and spreads between the cuticles and that a carbohydrate-containing substance is also found between the L2 and L3 cuticles throughout, at exactly the same sites in this nematode. This glycoprotein, which is either secreted from the mouth or the amphids or both is exuded whether or not the larvae have been stimulated to exsheath. Slight staining was also sometimes detected at the secretory-excretory pore and to an even lesser extent at the anus. This glycoprotein is thought to function as a lubricant to prevent abrasion between the cuticles. It accumulates in areas where there is greatest space between the cuticles such as the head, tail and where bending of the nematode body occurs.

Ultrastructure of the tail region of the second stage preparasitic larva of the root-knot nematode

Abstract Studies on the tail of second-stage infective larvae (L2s) of Meloidogyne javanica, M. incognita and M. hapla from a region anterior to the rectal gland to the tail tip have revealed the presence of a previously undescribed sensory organ, the caudal sensory organ, in the posterior region of the tail. The extreme tip of the tail consists of solid cuticle and the different zones of this structure are described throughout the tail region. Longitudinal sections through the anus and rectum have revealed that the cortical (external cortical, epicuticle) layer gradually decreases in thickness until its outermost layer appears to merge with the plasma membrane of the rectal inflation or gland. This gland appears to be similar in all three species studied. The two phasmidial glands and their canals are described from transverse sections. Somatic muscle is first found in the region of the anus and it extends anteriorly throughout most of the length of the L2. Depressor ani muscles which insert on the dorsal surface of the rectum are also described. The rectal gland in the dilated state occupies about three quarters of the diameter of the L2. It contains a matrix which resembles that extruded from the adult female rectal gland cells. The rectal gland cells contain large amounts of rough endoplasmic reticulum and desmosomes are found close to the junction of these cells and the plasma membrane of the gland itself. More anteriorly in the L2 most of the area is taken up by large lipid droplets which function as an energy reserve. It is suggested that the rectal gland should not be used as a taxonomic criterion for separating the La2S of Meloidogyne because it can vary so much in appearance within the same species.