General introduction and literature review
The purpose of this study has been to evaluate geographic variation of the nematofauna, to analyse important influences, and to determine the influence of climate, and how it works. The experimental part of this study is restricted to soil and plant nematodes with emphasis on the nematofaunas in Venezuela and in the Netherlands. The introduction and literature includes also freshwater and marine nematodes.
The extensive literature survey summarizes data about occurrence and main characteristics of nematodes. They are the most ubiquitous group of the animal kingdom, constituting more than 80 % of all multicellular animals (Table 1). Despite numerous inventories made for faunistic and agricultural purposes all over the world, pertinent knowledge about the geographic distribution of well defined species is scarce and data to explain the situation are fragmentary. This is due to incomplete coverage of the sampling areas and of the habitats within an area, to erratic collection and poor identification of the nematodes, and to the primitive state of study on nematode biology and physiology.
An attempt is made to review faunistic nematode studies in three sections. The initial surveys (1.2.1), from BORELLUS' discovery of the first freeliving nematode in 1656 to DE MAN'S classic faunistic studies in 1884, are discussed in detail. They aroused the interest of many zoologists and stimulated activity. The zoological surveys and expeditions (1.2.2), numerous from 1884 up to World war II and later more incidental, are fully recorded and briefly discussed where necessary, to obtain a near-complete review of this period. The results ascertained the widespread occurrence and the pluriformity of nematode communities in widely varying habitats and furnished some ecological observations on relation of nematodes to food, moisture, soil type and climate. The inventories for agricultural purposes (1.2.3), rapidly increasing in number up till now, are already far too numerous to be cited here. The relevant publications are listed in a separate publication (DAO et al
1970). The information is brought together in sections on general surveys per area (in which the areas covered are listed), general surveys per plant (in which the plants or plant groups studied are recorded), and surveys for a specific plant nematode or group of nematodes. The distribution pattern of the best studied species, Heterodera rostochiensis,
is mapped (Figure 1). The inventories for agricultural purposes reveal the widespread occurrence of dense populations of plant nematodes, omnipresent in all soils and all crops, and suggest strong influences of plant species, soil type and geographic situation.
The main environmental influences on a nematode population in the soil are listed and are synthesized into a qualitative scheme which is descriptive, functional and causal with respect to the relations between nematodes and their environment (Figure 2). Essential modifications from similar schemes for other animals are the acceptance of a direct influence of climate, soil and other physical and chemical components on nematode populations, and the omitting of active migration due to limited locomotion. The passive spread of nematodes, however, is extremely efficient. Together with their great persistence, their polyphagy and lack of interspecific competition, this potency may account for the widespread occurrence of polyspecific nematode mixtures in soil. Nematodes are normally carried passively but alive into a habitat, after which the environment becomes operative. They have to survive and reproduce before the population becomes measurable and can be related to the geographic environment. As soon as a population is measurable it is already certain that the habitat affords suitable conditions for survival and propagation of the species.
Climate, soil and plant factors are recorded as determinant for the development of nematode populations. Because it will appear later that soil, plant and other local factors cannot account for the geographic variation of the nematofauna, further attention is focussed on climate. The influence of climate and weather, average and momentary effects of atmospheric conditions, is usually reflected in the main density and the density fluctuation of a population. Most nematode infections carried into unsuitable climate and soil environments become extinct. It is stressed that most population-dynamic studies have been on existing, and therefore successful combinations of species and environment. KLOMP'S statement (1962) that climate or weather cannot regulate density directly, because too many populations would soon become extinct, is meant for such established, and therefore selected populations. Innumerable nematode inoculations, however, must become extinct due to the direct influence of climatic, soil and other environmental influences, immediately or after a number of generations. This direct influence may be called destruction or determination rather than regulation, but it must be a main governing process in determining the borders of the range of nematode species and it is therefore included in the scheme of Figure 2.
Temperature will appear as the main climatic factor related to geographic distribution. Therefore literature data about temperature are collected. Nematological thermograms show the normal type of biological optimum curve, demonstrating kill, cold stupor, limited activity, optimum, limited activity again, heat stupor and kill successively in a gradient from low to high temperature.
Complete data on any one species are not available, but bits of information on more than 140 different cases of a nematode's activity related to temperature could be listed (Table 2). Most soil and plant nematodes are active and thriving between 15° and 30°C and become motionless at 5°-15°C and at 30°-40°C.
Minimum temperatures for hatch and emergence, invasion and activity and optimum temperatures for reproduction and other processes are apparently low for plant parasites in temperate climates (e.g. Heterodera avenae, H. trifolii, H. rostochiensis, H. schachti, Ditylenchus dipsaci, Meloidogyne hapla),
whereas they are high for plant nematodes from warmer regions (e.g. Hemicycliophora arenaria, Tylenchulus semipenetrans, Trichodorus christiei, Scutellonema brachyurum, Heterodera glycines, Meloidogyne
and some other species). The maximum temperatures for activity, generally between 30° and 35°C, do not seem to be correlated with the geographic range of the nematodes. Temporary tolerance to extreme temperatures, and to drought, may be determinant for survival, and for the geographic range of a species, and diurnal and seasonal fluctuations may increase or decrease certain nematode activities and their susceptibility to extreme temperatures. The literature already suggests a strong influence of temperature on the distribution pattern of nematodes.Materials and methods
The materials and methods regularly used in the experimental work are described in Chapter 2. The faunistic studies and the evaluation of population densities in soil, plant tissues or other substrates were based on standard, known sampling techniques. The nematodes for faunistic and taxonomic studies were put into permanent slides and stored in the collection of the Landbouwhogeschool. The nematode populations for experimental work were some natural communities in their original soil, and also a number of selected species from Venezuela and the Netherlands reared and kept as monospecific populations in greenhouse compartments: Meloidogyne incognita
from Venezuela (V) and from the Netherlands (N), M.hapla
(V) and (N), Ditylenchus dipsaci
(V) and (N), Aphelenchus avenae
(V) and (N) and Helicotylenchus dihystera
(V). The main plants used for nematode propagation and for experiments were tomato, maize, phlox and bulbs of onion, tulip and narcissus, as well as agar cultures of Alternaria solani
. The soil was usually sterilized potting soil. Apart from clay pots and petri dishes the usual containers for nematode studies on plants were plastic tubes of 4 x 4 x 20 cm. Different facilities or apparatuses were used to control temperature, namely greenhouse compartments and climatic cells, Wisconsin tanks, a series thermostat and special incubators.
Estimation of nematode densities in soils, plant tissues and agar cultures was made according to methods described by OOSTENBRINK (1960) and s'JACOB & VAN BEZOOIJEN (1967), with some modifications for extraction of Aphelenchus avenae
from agar fungus cultures. Most experiments were replicated and the results were statistically treated, after transformation of the nematode and plant figures as indicated in the tables if this was considered desirable.Analysis of the nematofaunas in Venezuela and the Netherlands
Table 3 lists the nematode species recorded from Venezuela and from the Netherlands, 176 and 425 species respectively. These countries were chosen as test areas for comparison of their nematofaunas and for the selection of suitable test species because: a) the countries represent a tropical and a temperate climate, however with temperate conditions on the Venezuelan mountains and with tropical conditions in greenhouses in the Netherlands; b) the nematofaunas of both countries are fairly well studied; c) the nematodes of both these countries have largely been identified and could be checked at one place, namely the Landbouwhogeschool, Wageningen; d) the author was personally involved in nematode studies in both countries.
Table 3 shows that the nematode faunas of the two countries differ markedly. The difference is evident at the generic level and certainly in the species range of genera with many species. Scrutiny of the data reveals that of the 63 species reported from both countries only 21 may safely be considered to occur in the tropical and the temperate climate, whereas 7 were incorrectly identified for one country or the other, 13 appeared to be uncertain for poor state of taxonomy of the genus in question or for insufficient species diagnosis and 22 were found at high altitudes in Venezuela or in greenhouses in the Netherlands only. The 21 species which may live in both climatic zones, against at least 362 in the Netherlands only and 113 in Venezuela only, are microphagous or predatory, with only two possible plant parasites: Macroposthonia sphaerocephala
and Nothocriconema mutabile.
Ubiquitous species, therefore, are rare.Transmission and inoculation experiments
The differences in nematofauna cannot be related to the geographic situation of the two countries as such or be due to insufficient transportation of inoculum, for many tropical species are found in Dutch greenhouses and many temperate species on Venezuelan mountains. The known records of plant parasites are brought together in Table 4. Environmental factors must therefore be instrumental. The influence of climatic factors was studied in inoculation and transmission experiments in which the influence of soil, host and other organisms were excluded as far as possible.
Inoculation experiments at a lowland station of Maracaibo, followed by transport of half the pots to an altitude of 2800 m after two months, revealed that subsequently the lowland species Meloidogyne incognita, Pratylenchus zeae
and Helicotylenchus dihystera
developed better at 0 m and the mountain species M. hapla
developed better at 2800 m (Table 5). Aphelenchus avenae
developed at both altitudes in this experiment.
Transmission of a natural lowland nematode community in its original soil to the mountain and of a mountain community to the lowland, with control pots at the original locality, confirmed this result of the inoculation experiments. Most of the lowland species declined at high altitudes and the mountain species at low altitudes, except the composite group of saprozoic nematodes (Table 6).
Transplantation experiments with tropical Meloidogyne
species in the Netherlands revealed that these species cannot maintain themselves out of doors (4.4). Infested plant material planted outdoors is nematode-free after one to three seasons.
The direct influence of locality, soil, plants and other growth factors was excluded here. This leaves temperature, and for the experiments in Venezuela possibly air pressure, as instrumental influences which have to be studied further.Air pressure effects
Experiments in exsiccators showed, that the (V) and (N) populations of A. avenae
and D. dipsaci
and (N) did not reproduce less at a low air pressure equivalent to the situation at 3000 in above sea level than at the normal atmospheric air pressure at sea level (Tables 8 -11). Low air pressure as at high altitudes therefore does not determine the reproduction of these nematodes, and it is unlikely that it influences the geographic range of nematodes.
It was found in these experiments that the reproduction of A. avenae
on cultures of Alternaria solani
and of Ditylenchus dipsaci
in onion bulbs were both suppressed when too many fungal cultures or too many onion bulbs were placed together in a closed exsiccator. The fungus grew poorly and the onion bulbs decayed. It is probable that these materials released nematicidal agents, which could not be fully identified by gas chromatography.Temperature effects
It appeared from the Chapters 3, 4 and 5 that climatic influences on nematode communities are great, and by exclusion of the other influences the conclusion seems justified that temperature is the key factor. Therefore the influence of temperature ranges and gradients on biological activities of one or more populations of six nematode species was extensively studied.Meloidogyne incognita.
Comparative experiments with the (V) and (N) populations were made on hatching (Figures 3 and 4, Table 12), on penetration, reproduction and gall formation after inoculation onto tomato plants in greenhouse compartments (Tables 13 and 14, Figure 5), in Wisconsin tanks (Tables 15 and 16, Figure 6) and in climatic cells (Tables 17 and 18, Figure 7), and about the morphology (Figure 8, Table 19). Both populations appear to be thermophil. Hatching, gall formation and reproduction was abundant and rapid at 25° and 30°C and for the (N) population also at 20°C. They were less at 15 OC, except for nematodes that had already penetrated at a higher temperature, and they dropped to a low value at 10°C. A 15-day exposure of eggs in water at 5° or 35°-40°C killed more than 80% of both populations (Table 12). The temperature of 35°C was too high for the eggs in water, but galling and reproduction on tomato occurred at 35°C, probably because a growing plant is cooler than the surrounding soil at high temperatures. The (V) and (N) populations were morphologically the same, but the (N) population was somewhat more thermophob and markedly less cryophob than the (V) populations which may be significant for the geographic range of the nematodes. All data together indicate that the minimum temperature for the (N) populations to start infection and reproduction is about l0°C which is about 5°C lower than for the (V) populations, and also that the larvae of the (N) population inoculated in soil may survive 15 °C for at least 4 weeks whereas the (V) population cannot survive that period. It therefore apears that there are strains with different temperature requirements in the morphological species M. incognita
which were still present after propagation of the nematodes on the same plant in the same environment for more than one year. We propose to indicate strains with a fixed difference in temperature requirements as thermotypes.Meloidogyne hapla.
Experiments similar to those described for M. incognita
were made with the (V) and (N) populations of M. hapla,
and one experiment was added on survival at low temperature (Figures 9-13, Table 20-29). Both M. hapla
populations appear more resistant to low temperature than M.incognita.
Their survival was good at 5°C in water (Table 20) and at 0°C in soil whereas there was partial survival in soil at-10°C for several days (Tables 27 and 28). Both populations could start some activity at 10°C, but for the rest they were thermophil and could thrive at 30° and 35°C like M. incognita.
The fact that M.hapla is
widespread out of doors in temperate climates is therefore explained, for the species can thrive at low temperatures and can survive spells of low temperature, but unexplained is the fact that it is rarer in tropical regions and in greenhouses than M.incognita,
for its thermopreferendumis as high or higher. The two M. hapla
strains differ in morphology (Table 29) and in development of males (males at 35°C only in the (V) population but not at all in the (N) population) and there were slight differences in their temperature requirements. The (N) population appeared somewhat more resistant to adverse low and high temperatures and was somewhat more thermophil in its biological activities than the (V) population. These differences are small compared to the differences between M.hapla
and M. incognita.Aphelenchus avenae.
Experiments were in temperature ranges and gradients with the (V) and (N) populations grown on agar plates with Alternaria solani.
They were on reproduction (Figures 14 and 15, Tables 30 and 3 1), adaptation to lower temperature (Figure 16), sex ratio (Tables 32 and 33), influence of contaminating fungi (Figure 17) and morphology (Figures 18 and 19, Tables 34). Both populations are apparently thermophil; the differences between the two populations in several biological and morphological aspects, however, suggest that they may not be the same species. Reproduction occurs from 16°-35°C in the (V) and probably from 5°- 30°C in the (N) population, with an optimum for (V) slightly above and for (N) slightly below 25°C. The cryophob (V) population gradually reproduced somewhat better, although with temporary depressions, when grown for 24 months at 18°C (Figure 16) without losing its potency to reproduce strongly at 25°C. Males are absent at 25°C, but a less numerous population consisted mainly of males when populations were grown at 32°-33°C. Temperature appears to determine the formation of males in A. avenae
and this holds for both populations (Table 32). The influence of temperature is probably direct and not indirect via the fungus (Table 33). The main contaminating fungus in the A. avenae
cultures on Alternaria
solani was Trichoderma koningi,
which released a nematicide into the cultures (Figure 17). Temperature influenced the morphology of A. avenae
in all eight characters studied (Figure 18) and there were significant differences between the (V) and (N) populations at one or more temperatures for all morphological characters studied (Table 34, Figure 19). The two populations are probably different species. BERGMAN'S climatic rule that vertebrates of the same species grow bigger when they live at lower temperatures does not apparently hold for nematodes.Ditylenchus dipsaci.
Several experiments were made in temperature ranges or gradients with (V) garlic and with (N) onion, (N) tulip and (N) narcissus populations. The experiments were on reproduction in onion bulbs and phlox plants (Figures 20-27), on survival and conditioning of the narcissus and tulip strains in hot water (Tables 35 - 37), and on morphometric variation (Table 38, Figure 28). The optimum temperatures for infection, reproduction and other activities of D.dipsaci
are generally between 10° and 20 °C (Table 2) but it appears that populations differ significantly. The (N) onion population, grown in onion bulbs and in phlox plants, was less thermophil than the (V) garlic population although the last mentioned population is restricted to high altitude in Venezuela. Temperatures of 15°-20°C were favourable for both populations, but (N) onion thrived relatively better at 10°-15°C, and (V) onion was more numerous at 25-30°C; in fact (V) garlic showed an aberrant strong increase at 30°C in phlox (Figures 21 and 23). (N) tulip and (N) narcissus showed a relatively broad temperature spectrum with the same optimum of 20°C for reproduction; they are apparently more thermophil than(N)onion. Although the difference is small, (N) tulip appeared to be significantly more resistant to destruction by heat in water than (N) narcissus. Nematodes of both populations grown at the high temperature of 25°-30°C were more resistant to heat than populations grown at 10°-15°C; this difference was maintained for some weeks when the nematodes were stored at 3°C (6.5.5, Table 35). The (N) tulip population was also more resistant to heat after a pre-treatment at 35°C for 1-16 hours. This resistance is lost again when the nematodes are kept at 20°C for 14 hours, but not at 3 °C (6.5.7, Table 37). It is postulated that this temporary resistance induced by high rearing temperature or by pre-treatment at 35 °C is associated with a chemical principle. It was further noticed that nematodes may be motile after certain heat treatments, but nevertheless must be damaged to such an extent that they cannot stay alive and reproduce in their hosts (6.5.6). The data collected may help in understanding and in improving the results of commercial treatment of flower bulbs with hot water.
Some morphological characters showed differences between the (V) garlic and (N) onion populations at one of the two rearing temperatures but the characters influenced and the influence itself varies. And there are characters which did not show differences at all, either for population or for temperature (Table 38, Figure 28). This makes it unlikely that the two populations (V) garlic and (N) onion are different species although the significant differences noticed in some morphological characters and in the temperature requirements suggest that the possibility should not be excluded. Crossing between nematodes of the two populations are needed to decide this.Helicotylenchus dihystera.
The influence of temperature on reproduction of a tropical (V) population on tomato was studied in inoculation experiments in greenhouse compartments and Wisconsin tanks (Tables 39 and 40, Figure 29). Reproduction occurred at all temperatures tested in the range from 15° to 25° or 35°C in the greenhouse and Wisconsin tanks respectively. The population density, in the soil and in the roots, increased linearly with temperature up to the highest temperatures, 25° and 35°C in the experiments mentioned. H. dihystera
therefore is a tropical nematode, although it may perhaps thrive in somewhat cooler regions.Pratylenchus crenatus
(N) and associates. Soil with a natural community of Pratylenchus crenatus, Tylenchorhynchus dubius
and Rotylenchus robustus
was grown with maize at different temperatures in greenhouse compartments and Wisconsin tanks (Table 41 A and B). The population of all three nematodes were optimum at 10°-15°C; and declined at the higher temperatures, although root growth increased much with temperature. After 98 days the populations at 25°-30°C were normally half to one twentieth of the original populations or of the populations at 10°C.
Treatment of a natural (N) nematode community in moist sandy clay soil without a host plant at different temperatures for periods up to 3,1 months yielded interesting results (Figure 30). All phytophagous Tylenchida were suppressed from 20°C upwards and they were eradicated at 25°-30°C or higher within 3½ months. Temperature apparently has a determinant influence on the course of plant nematode infections taken in soil from temperate zones into tropical regions; the same may hold for tropical nematodes taken to a temperate climate. The saprozoic nematodes as a group were less susceptible to heat than the plant nematodes. These data are in accordance with the faunistic data of Chapter 2 which indicate that the few possible ubiquists are usually saprozoics.Conclusion
The nematofaunas of the two climatic zones, tropical Venezuela and the Netherlands, differ conspicuously in the species. The few possible ubiquists found in both climatic zones do not comprise known or suspected plant parasites except two criconematid species. The fact that many tropical plant nematodes are common in Dutch greenhouses and temperate species are found in Venezuelan mountains indicates the far spread of inoculum. The results may be of value for plant quarantine.
Experimental exclusion of the direct influence of locality, soil, plant and other growth factors shows that the distribution range of nematodes must be determined to a large extent by climate, especially by temperature, as could be expected from the literature.
Effects of temperature on survival and thrift of a nematode differs markedly between species and between populations of the same species. The climatic or geographic range of a nematode appears closely related to lethal temperatures, or optimum temperatures for activity, or diverse combinations.
Temperatures of 25 °C and higher were lethal to plant nematodes in implanted soil from the temperate zone, and temperatures of 5°C or lower were lethal to nematodes from the tropical zone within ½-3½ months. This excludes hibernation in the other climate in the absence of growing hosts. Also established populations on growing host plants placed in the other unsuitable climate decline or disappear, although this takes apparently much longer than in the absence of hosts.
The temperature influences found to be instrumental for the populations studied are summarized in Table 42. The populations of the tropical zone do not start their activity under 15°C and thrive at 25°-35°C, e.g. M.incognita (V), A. avenae (V)
and H. dihystera
(V). M. incognita
(N) from Dutch greenhouses is intermediate. The nematodes from the temperate climate, the Netherlands and the Venezuelan mountains, survive spells of very low temperature and become active at about 10°C. The temperature range at which they thrive varies; it may be low, i.e. 10°- 20°C for D. dipsaci
(N) and P. crenatus
(N), moderate i.e. 15°-30°C for A. avenae
(N) and D. dipsaci
(V), or even as high as for the tropical species, i.e. 20°-35°C for M. hapla
and (N). M. hapla
(N) and (V) do not differ from M. incognita
(N) except for the fact that the last species cannot survive very low temperatures. The populations which were studied appear to be well temperature-adapted to their climate, but the mechanisms vary.
In the same morphological species the temperature requirements of populations differ. These differences are stable and therefore probably genetic (thermotypes). Some adaptation or selection occurred when A. avenae
was grown at a sub- lethal low temperature for 24 months. Rearing and storage temperatures induced a principle for heat resistance in D. dipsaci
which was not stable and may be of chemical nature. At the other hand temperature influenced the morphology of A. avenae, D. dipsaci
and M. hapla
(with maximum size specimens at the optimum temperatures) and determined the formation of males in A. avenae).
All data together illustrate the value of the morphological species to characterize the difference between nematofauna and between individual species approximately. The profound influence of temperature on morphology and the marked physiological specialisation within the species studied, however, underline the great value of the biological species concept in work with nematodes.