1. Useful background knowledge for legume N fixation research in a cold grassland area like Xiaman
Abstract: This introduction covers physiology, the Xiaman
experimental site, N fixation ecology and research methods. Its aim
is a) to explain basic ideas to a reader with a background in
general biology and b) to give access to recent literature in the
fields concerned.
1.1. Physiology of general N fixation and the legume-rhizobium symbiosis
1.1.1. Evolution and energetics
Nitrogen, element No. 7, is an essential element for all forms
of life so far discovered on our planet, it is for example part of
all proteins and nucleic acids (the carriers of genetic
information). Unfortunately, in the oxidative atmosphere prevalent
since ca. 2 billion years, most nitrogen has not been readily
available to most organisms, but rather been concentrated as
dinitrogen gas in the atmosphere. Although the covalent N-N bond is
rather stable, a number of organisms, scattered throughout the
kingdoms of the Eu- and Archaebacteria (#Woese 1987), have parallely acquired
the ability to fix nitrogen, maybe relatively lately in evolution
by horizontal transfer of genetic information(#Postgate 1992). Nitrogen fixation,
i.e. the transformation of atmospheric nitrogen into hydrogen- or
ultimately carbon-bound nitrogen which all organisms can use,
proceeds via enzymes called nitrogenases (quite a misnomer), the
entire structure of the most efficient molybdenum variety believed
to have been uncovered recently. It consists of different Fe/S
clusters, an ATP-reaction site and an active Mo site for dinitrogen
binding and reduction. As the molybdenum center is activated by
hydride formation, one necessary side-product of the reaction is
hydrogen (possibly protecting the oxidation-sensitive enzyme). In
legumes, special hydrogen recycling (uptake) systems exist. It
should be noted that nitrogenases with non-molybdenum coenzymes
such as vanadium-iron in the azotobacteria are considered to be
less efficient because they release huge amounts of hydrogen (#Frausto 1993:426).
Of course, this ingenious way of capturing atmospheric nitrogen
is not free, but rather energy-intensive. #Rao(1987:247) reviews estimates between
1.26-1.6 mol glucose consumed per mol of nitrogen fixed into carbon
skeletons. However, this doesn't look that horrible when compared
to the costs of nitrate incorporation (1.4-1.5 mol glucose consumed
per mol of nitrogen fixed) or even ammonia assimilation (also
consuming 0.7-0.8 mol glucose, the bulk of it used for carbon
skeleton incorporation). Or if I look at it from a monetary
viewpoint, an estimated 150-175 billion kg annual biological
nitrogen fixation worldwide (Rao, ibd.) equal ca. 1.2-1.4 trillion
(US $ 160-180 billion) Yuan of equally if calculated after Chengdu
market prices for urea fertilizer.
Being so useful, nitrogen-fixing microorganisms often happen to
end up in more or less close symbioses, such as the Frankia
actinomycete with many (especially woody) plants (#Benson 1993#Forestry Soil Institute of the Academia
Sinica 1985#Deng 1992) or
cyanobacteria in lichens or with the tiny Azolla on rice paddies or
azotobacter in the cow rumen. The best-investigated of these
communities is the legume-Rhizobium symbiosis, which shall be
briefly introduced below.
1.1.2. Particulars of the legume-rhizobia system
Legumes ("bean plants"/Fabaceae) are a family or superfamily of dicotyledonous plants, in species diversity they are third most large family of plants (ca. 5% of Earth's plant biodiversity, after the Poaceae("grasses") (7%) and Orchidaceae(7%)). It may be safely guessed that they make up for more than at least for 1% of the total Earth plant biomass, and they comprise well-known crops such as soybeans, mung beans, faba beans, French beans, peanuts and peas as well as forages such as clover, sainfoin, fenugreek and vetches which ranks them second in importance of nutrition for mankind (after the Poaceae; see #Allen 1981for extensive account; for a database (#Bisby 1996) visit www.soton.ac.uk/~ildis). Although during the "Green Revolution" advances in legumes breeding haven't been so big as in the Poaceae (definitely mankind's most important nutrient source for the last five millennia), they are now being reappraised as a useful tool for ecological agriculture (#Giller 1996).
The soil is probably the most abundant habitat for microorganisms, and this is also the place where rhizobia do thrive (although they tend to be present in low numbers). For bacteria doing such energy-intensive jobs as nitrogen fixation, the area around plant roots ("rhizosphere", #Yan 1993 #Doebereiner 1988) is particularly attractive. So it doesn't come as a surprise that most rhizobia preferentially interact with the legume("bean-like plant",see below) root, and this is the habitat their name is derived from. From their phylogenetic relationships (being in the alpha-proteobacteriacea/alpha-purple bacteria, #Stackebrandt 1988) it is evident that, similar to mitochondria, phyllobacteria and agrobacteria, they have been a plant pathogen, but one which has been tolerated due to its usefulness for the host plant.
The symbiosis is confined to a special organ called nodule
(meaning: "little knot") in most Western languages, referring to
the typical knot structures visible on most temperate grain legumes
in Europe (such as peas). Interestingly, the retranslated Chinese
idiom ("genliu") means "root ulcer" which corresponds to the more
ulcer-like form on Asia's dominant grain legume, the soybean. When
dwelling on East-Western nodulology, it might be added that there
is a vague speculation that the ancient Chinese character for
"bean" included three dots indicating nodules (#Hymowitz 1970 #Bergersen 1980), but this theory
which would antedate the first Western documentation (Fuchs 1542)
by more than two millennia doesn't seem to find much credit in
China (try to look up the Hymowitz paper in a mainland
library).
For a long time, rhizobia have been believed to be able to fix
nitrogen only in the nodules, and it was as late as 1975, that #Pagan demonstrated rhizobial N
fixation on an N-free and legume-free medium. Although more recent
findings (such as #Dreyfus
1983) affirmed that the capability of independent N fixation is
widespread, it is still believed that the bulk of rhizobial N
fixation occurs in the more eloborate nodule system.
1.1.2.2. Different legumes often need different rhizobia
19th-century microbe hunters can be credited to have classified
nodule organisms even before anybody opined they might be useful
(#Schroeter 1886, see #Moffett 1968). Within a decade of the
discovery of the nitrogen-fixing properties of the symbiosis (the
classic paper is #Hellriegel
and Willfarth 1888), it was established that not every kind of
bacterium will be able to "nodulate" (i.e. to form little knot-like
structures on) every plant. This led to the formulation of a dozen
of "cross-inoculation groups" (#Mueller 1925#Fred 1932), these are groups of strains
which are capable of replacing each intragroup strain in order to
form symbioses with a certain group of legumes, but strains from
different groups are unable to replace each other. For examples,
vetches and peas form such a group, and if you isolate a strain
from faba bean, you might expect it to nodulate the green manure
vetch Vicia sativa, but not soybean which is in a different group.
To give another analogy, Chinese people are able to read Chinese
books (regardless of published in Harbin or Haikou), Japanese are
able to read all material published between Hokkaido and Okinawa,
and so on. But you will note that this correlation is not a perfect
one: quite a few Chinese know Japanese and vice-versa. Furthermore,
in China, there are millions and in Japan there are thousands whose
mother tongue isn't Chinese or Japanese at all. So this relation
(although of great practical value) is a limited one, and the same
is true for rhizobial inoculation groups, some groups are rather
well-defined (such as the above-mentioned vetches or medics) and
others are indeed quite loose; however there is no need to be as
cynical as #Wilson (1944) who
polemically termed his paper on astragalus rhizobia "Over 500
reasons for abandoning the cross-inoculation groups of
legumes".
1.1.2.3. How different legumes recognize different rhizobia
Although - in great contrast to section 1.1.2.2. - the details
of the plant-rhizobium interaction are not at all crucial for the
understanding of this thesis, this fascinating model for symbiotic
interspecies communication deserves to be introduced - the major
steps of the most common pathway are:(1)If there is a need for
nitrogen (#Coronado 1996),
the legume produces flavonoids and excretes them to the
rhizosphere.(2)Water-soluble flavonoids travel through the soil,
rhizobia meeting them can dissolve them by C-ring fission(#Rao and Cooper 1994).By a yet unknown
mechanism, a nodD gene is activated by flavonoids to produce a NodD
protein activating other nod ("nodulation") genes (#Spaink 1994),(3)the most important
being nodABC(all species of rhizobia so far investigated have
them,#Tan and Chen 1992). NodABC
can produce the skeleton of the Nod factor eliciting nodules. The
nucleotide sequence of NodC resembles a chitin polymerase, and
possibly catalyzes N-acetyl-D-glucosamine polymerization. After
tetramers or pentamers of chitin are built, NodB removes the apical
N-acetyl-glucosamine residue, after that NodA catalyzes the
addition of a longer ester(#Denarie 1993).Although rhizobia can
excrete Nod factor into the medium, from its chemical structure it
a position on the cell membrane seems more likely(#Hirsch 1992).The chemical structure
of the Nod factor of each kind of rhizobia is different, broad
host-range rhizobia (such as NGR 234) have several Nod factors (#Franssen 1995). The first nod
factor whose structure has been cleared up was that of alfalfa(#Lerouge 1990).Except for the
common nodABCD genes, many nodulation genes are species specific,
e.g. alfalfa's NodH, NodP and NodQ are responsible for the
sulfurylation of the Nod factor;NodE,NodF and NodL participate in
the fatty acid synthesis(#Caetano-Anolles and Gresshoff 1991).
Alfalfa rhizobia lacking NodH have a Nod factor which is not
sulfurylated,thus they cannot nodulate medics, but are able to
nodulate vetches and peas(#Horvath 1986 #Denarie and Cullimore 1993).
When the rhizobial Nod factor comes to the plant root in nano- or picomolar concentrations,the classical reaction is:(4)first a calcium protein (rhicadhesin,#Smit 1987) shared by all rhizobia and agrobacteria mediates binding to the plant receptor having an Arg-Gly-Asp domain,possibly a lectin(#Swart1994) . For example, Arg-Gly-Asp is also distributed in a lectin called discoidin (#Barondes 1988). Of course, rhizobial binding is also influenced by environmental factors, such as pH,Ca,Mg and phosphate availability (#Caetano-Anolles 1989#Howieson 1993). (5) After 6-18 hrs, the fine root hairs of rhizobia begin to curl(#Hirsch 1992),(6)the infection threads are formed in the root hair(7)Rhizobia penetrate via the infection thread to the root cortex, and induce the cortex (inner cortex for indeterminate nodules,outer cortex for determinate nodules) to produce a meristem. Some researches think this step requires rhizobial exopolysaccharides, but this is still unclear.(8)Controlled by auxins, this meristem produces nodules( #Hirsch 1989),a new organ. In the well-researched alfalfa, this can be divided into an apical meristem, a medium zone containing rhizobia-containing plant cells (very inappropriately named "bacteroids"), and basal vasculae (tubes) maintaining contact with the plant metabolism.(9)For aeriation, the plant produces leghemoglobin and several other enzymes(#Caetano-Anolles and Gresshoff 1991).(10)The host plant can control the total amount of nodulation:#Nutman(1952)discovered that if you cut away the nodules of red clover,it is able to regenerate nodules, but will not form new nodules without cutting. This phenomenon has been discovered on quite a few plants, but its molecular biology is still unclear.(11)The nodules of some plants are shed in winter, some appear to be perennial. There has been little research on the mechanisms of this.
1.2. The Xiaman experimental site
1.2.1. Location on the Qinghai-Tibet plateau
The Qinghai-Tibet plateau (comprising nearly all of Tibet, whole
Qinghai, parts of Xinjiang, Gansu and Sichuan and the alpine
regions of most adjacent Himalayan states) is the hugest high
plateau of the world, and being the cause of the monsoon winds it
has profound influences on the climate of adjacent South East Asia.
But although a unit geologically and culturally, due to different
moisture belts, its vegetation should be divided into subunits
ranging from coniferous forests in the southeast to desert belts in
the northwest. Our research area is located on the Naqu-Yushu
alpine-semihumid belt (which is a bit colder and moister than the
better-known Lhasa shrub grasslands), which has been characterized
by #Zheng (1979) as
following:
"This area is located in the center and east of the plateau, it
extends from the east of the Nujiang (Mekong) via Yushu, Guoluo to
Ruoergai in NW Sichuan. The relief is rather shallow, there are
broad valleys, basins and hills, the altitude is about
4,000-4,600m, in the eastern Ruoergai down to 3,500m a.s.l. The
glacial relief is developed, and there are islets of frozen soil
surviving. The mean temperature in the warmest month is 6-10(12)oC,
the annual precipitation 400-700mm, dryness 0.8-1.5, in summer
hailing is common, and snowfall in winter is not negligible. In the
high alpine meadows and shrublands Kobresia, Polygonum, Salix and
Rhododendron are dominant, and create a high alpine felt soil.
Pedogenesis is characterized by large accumulation of humus, little
washing out, and long-time glaciation. As the grass layer is quite
hard, solifluction processes such as landslides and debris flows
are common. River systems are developed and peat soils do often
form."
Most of the plateau is used for cattle grazing by Tibetan nomads
(for an English introduction see #Miller 1990), and it is claimed that
since the reprivatisation of grazing systems overgrazing has led to
deterioration in some areas (#Xu
1990): For example, in Qinghai province there area per sheep
unit has been estimated to drop from 1.8 ha to 0.7 ha in 1985; meat
weight dropped by 30% per individual and the whole productivity of
wild grasslands dropped by 30-60% and with an desertification rate
of 1.8% p.a.(#Shi 1992). The
detrimental effects of overgrazing have also been shown by #Han et al.(1991) who reported an
increase in forbs from 21.7% to 61.6% and an increase in poisonous
plant biomass from 0.57% to 1.39% when cattle density was increased
from 2.14 to 6.07/ha.
Furthermore, although to date relatively unspoiled by pollution,
the plateau might suffer from global warming; a joint State
Meteorology Administration (SMA) - WWF study predicts this
environment to shrink about 28% to the middle of the next century
(this is a 1992 estimate, see #Feng
and Wang 1996).
In the eastern part of the Qinghai-Tibet plateau, the
characteristic peatlands in the provinces of Tibet (#Zhao et al.
1982), Gansu (#Yun et al. 1994), and especially NW Sichuan (#Qi
1960,#Chai et al. 1963,1965,#Zu 1983,#Yang 1986,#Sun et al.
1987,#Tsuyuzaki 1990,#Tsuyuzaki 1992, #Bjoerk 1993 #Yang and Jin 1993B, #Zhao1995 and 1996) have attracted much
scientific attention, but data on other vegetation types (such as
the subalpine hill vegetation) is more scarce (#Liu 1984#Ni and Wei 1984#Yang 1987#Chen 1992#Zhang 1994 #Liu 1994). The most exhaustive account
on Xiaman vegetation has been given by #Wei Taichang and Zhao Zuocheng (1986), a
species list of vascular plants and vertebrates can be found in the
appendix (on disk) of this work.
The Ruoergai high plateau (sometimes included in the "Songpan
grassland") is a plateau surrounded by mountains of ca. 4000m
a.s.l., extends over 200km NS direction and 100km EW direction, its
average elevation is more than 3,400m. The rock formations are
rather simple, mainly consisting of Triassic graygreen or yellow
sandstone (most of it metamorphosed to slates and phyllites) and
black shales. The relative height of the surrounding mountains is
300-500m, but the hill height inside the area is usually 70-150m,
sometimes up to 200-300m and running parallel. Most SE slopes are
rather steep and cirques rather complete, NW slopes are more
genteel. River valleys are very broad (up to 18km) and have many
sediments, at areas where waterflow is sluggish marshlands form
(#Chai et al. 1965).
Although in winter there is not much precipitation, the area is
moister than most of the middle and western parts of the plateau:
in the semiarid winter climate changes to semihumid in May and
thanks to Indian and South China sea monsoon it humid from June to
October; so according to the Penman formula its aridity is less
than 1 (0.93) and belongs to the humid zone (#Qian and Lin 1965), so the turning
green and wilting times are earlier than in the rest of the
grassland (#Zhang Yiguang
1985). Inside the Ruoergai grassland, the north is more
slightly more arid than the south, data for Longriba are 753mm (#Chai and Jin 1963), Hongyuan 613mm
(#Qin et al. 1985), Tangke 647mm
(#Chai and Jin 1963), Ruoergai
657mm (#Ministry of Civil
Affairs 1993), Maqu 616mm (#Yun
1994).
Mean annual temperature data are much more uniform, viz. 1.0oC
for Longriba (#Chai and Jin
1963), 1.1oC for Hongyuan (#Qin et
al. 1985), 0.8oC for Tangke (#Chai and Jin 1963), 0.9oC for Ruoergai
(#Ministry of Civil Affairs
1993), 1.1oC for Maqu (#Yun
1994). Most published data available is on Hongyuan, its medium
temperature in July is 10.9oC, diurnal temperature amplitude is
16.1oC, annual solar irradiation is 2147 hrs., in the growing
season photsynthetically active irradiation is 31000 J/cm2*month
during May-Aug and 22000 J/cm2*month during Sept-Oct,
photosynthetically fixed energy is less than 1% and biomass
produced yearly is 2000-3000kg/ha in most grasslands. Average peat
soil temperatures at 5 cm depth are 5.9oC in May, 13.6oC in July,
9.3oC in September. From end of October to end of April the soil
freezes, reaching a maximum ice depth of 40cm (marshlands) and 60cm
(grasslands) in February (#Yang and
Jin 1993).
Soils are mostly influenced by water availability and according
to the "diagnostic" Chinese soil classification system (#Gao
1990;note that there is some discussion to modernize the soil
classification system,see #Wang (1994)) can be classified into
three forms (#Chai et al. 1965):
(I)Organic soils: peat soils: mainly distributed in the broad
river valley of Heihe and partly distributed in Baihe river valley,
continuously, seasonally or temporary waterlogging occurs. The peat
layer usually is more than 3m thick and can occasionally reach 6m.
These soils are very rich in organic matter (more than 50%), total
nitrogen (5-8%), the pH is 7.0-7.8. The communities (I.I) and
(I.II) have similar nutrient dynamics, crude protein is highest in
June, biomass highest in July/August (1500 and 1650kg resp.).
(I.I)Marshlands always moist: Equisetum limosum, Potamogetum,
Hippuris vulgaris, Myriophyllum spicatum,Cicuta virosa, Menyanthes
trifoliata, Glyceria aquatica, Polygonum aquaticum,Triglochin
maritimum, Carex meyeriana, Utricularia media etc.
(I.II)Marshlands seasonally moist or intermediate stages:Carex
muliensis, Carex capillifolia, Sanguisorba filiformis, Caltha
scaposa, Aster alpinus, Deschampsia caespitosa, Blysmus
sinocompressus,Gentiana sino- ornata, Chamaesium paradoxum,
Ranunculus longicaulis/pulchellus,Trollius ranunculoides, Kobresia
humilis/parva/tibetica, Parnassia trinervis, Koeleria cristata,
Juncus leucanthus,J.concinnus,Pedicularis resupinatus,P.
rhinanthoides, Ranunculus brotherusii var.tanguticus, Anemone
obtusiloba, Eleocharis valeculosa, Koeleria cristata,Poa
chalarantha,Elymus nutans (#Bjoerk 1993). In case of overgrazing,
the tall grasses (such as Poa, Elymus) will become less and the
cyperaceae will dominate. In areas disturbed by rats, tuber root
plants such as Blysmus sinocompressus,Carex enervis,Potentilla
anserina will have an advantage.
(II) Alpine brown soils (or: alpine yellow-brown lime soils,
("Sichuan Land Resources Map Collection" 1990), sandy grassland
mixed soils ("Hongyuan Soils" 1985). On the dry river banks of Bai
river (such as Tangke) and the Reerba in Ruoergai. The matrix is
yellow sediment soil, the upper 0-4cm are close root cover, then
follows are brown-black humus layer (30-40cm thick), with an
organic matter content of 3-5%, it has carbonic acid inlayers (up
to 10%), pH 6.0-7.5, below 40cm it becomes sandy, sometimes there
are small dots of ferrous inclusion. This soil is the most fertile
in the area.
(II.I) The Xiaman river plain sandy soil environment has been characterized by Liu (1994) who listed Elymus nutans, Elymus sibiricus, Geranium pylzowianum, Kobresia capillifolia (=maquensis), Anemone rivularis and other plants.
(II.II)In overgrazed grasslands(plant height 10-30cm)the
dominant plants are: Stellera chamaejasme, Artemisia hedinii,
Aconitum carmichaeli, Pedicularis remitorta, P.oederi, Elsholtzia
densa, Eruca sativa (#Yang Dingguo
1987). In dry areas, Stipa spp. will dominate.
(III)Subalpine felt soils(also called: "subalpine grassland
soils"; "histosols" on the 1974 FAO World Soil Map):Distributed on
the surrounding subalpine mountains and hillocks (3420-4000M;
alpine grassland soils in the highest points). In contrast to
category (II), the matrix is sandstone shale or slate. The
grassroots layer is several cm, the brown-black humus layer 20-30cm
deep and organic matter content reaches 5-15%, pH is neutral.
(III.I)Here we find typical subalpine "Five-Flower-Meadow"/ forb
meadows with a height of 30-80CM,representative are :Kobresia
setchwanensis, K. capilliforma, Elymus nutans,Roegneria nutans,
Koleria litwinowii, Helitrotrichon tibeticum,Brachypodium
sylvaticum,Agrostis spp., Carex moorcroftii,Carex filipes, Stipa
aliena, Polygonum viviparum, P. amatum, P. avicluare, P.sibiricum,
Saussurea graminea, Anaphalis hancockii, Anaphalis lactea,Swertia
franchetiana, Gentiana timensis, G. straminea,
Ranunculuspedicularis, Festuca ovina, Clinelymus sibiricus,
Trollius ranunculoides, Leontopodium longifolium, Allium cyaneum,
Scrofella chinensis, Coluria longifolia (#Liu Qi 1984 specimen by Zhang
Zhaoqing).
(III.II)On southern slopes, xerophilous plants such as
Anaphalis,Stipa spp., vertches. Kobresia spp. will become more
dominant.
(III.III)On northern slopes, typical shrubs include Spiraea
schneideriana var. amphidoxa, Potentilla fruticosa, Spiraea alpina,
Lonicera tibetica. Above 3800m Rhododendron violaceum can be found.
Herbaceous plants to be found includeHelictotrichon tibeticum,
Festuca rubra, F. ovina, Elymus nutans,Brachyelytrum erectum, Poa
annua, Koeleria cristata, Ptilagrostis dichotoma, Deyeuxia
scobrescens, Carex digyna,Polygonum viviparum, P.sphaerostachyum,
Pyrethrum tatsiense, Hedysarum sikkimense,Meconopsisspp.(#Liu Qi 1984). On degraded slopes, these
are replaced by shrubs like Caragana
tibetica,C.erinacea(height:45-50CM)and herbs like Elsholtzia
fruticosa, Festuca aliena, Kobresia pygmaea,Saussurea
sp.,Potentilla multicaulis, Poa annua and Iris spp.(#Chen Quangong 1992).
(III.IV)On moist riverbanks Rhododendron violaceum, Artemisia
spp. , Salix spp.,Hippophae can be found.
(III.V)On the upper levels of the surrounding mountains, from
3800-4000M to 5200M alpine felt soilswill become prevalent and
their typical vegetation includes Kobresia pygmaica, Poa
sinattenuata var.vivipara, Polygonum viviparum, Coluria
longifolia,Spenceria ramalana, Anemone geum, Anaphalis flavescens,
Leontopodium longifolium, Saussurea stella, Arenaria sp.,
Androsacetapete etc.(#Liu Qi
1984).
(IV) Parallel to the yellow river, dunes can be found. They have
increased in height from 2-3m since the 60s (#Chai 1965) to 5-10m, their plant cover
is scarce, however rich in legumes. See section 2.3.5. for
discussion.
1.3. Ecology of leguminous nitrogen fixation
Rhizobia are relatively scarce, their number seldom exceeds 1%
of the soil population. If let alone in the soil, their numbers
decrease (#Cao 1994): they are rhizosphere organisms highly
stimulated by the presence of plant roots, in the short term these
stimulating plant roots are not necessarily legumes, on the long
term however presence of a certain host certainly enhances the
presence of appropriate inoculation group rhizobia: #Mahler (1982) reported a
rhizobia-other bacteria ratio of 1/50000 is non-cultivated and
1/500 in cultivated alfalfa fields. In a tropical soybean-rice
rotation environment, #Simanungkalit (1995) found
population density changes of 1-3 magnitudes between plant
cultivation and fallow periods, and these changes apparently do not
rely very much on which plant is cultivated: for example, he counts
72 rhizobia/g soil in a fallow lowland rice field and 26000
rhizobia/g for lowland fields cultivated with rice. In a temperate
environment, #Wiehe (1995)
showed that marked rhizobia grew nearly as well as pseudomonads in
maize, wheat and rape rhizospheres. He also reported that during
one growth period rhizobia were able to colonize non-legume
rhizospheres as far as 0.6m apart from an original site of
inoculation.
1.3.2. Habitat-dependent efficiency: crops and pastures
In crop environments legumes are either used as cash crops or as
green manure fertilizers. Although green manures such as Astragalus
sinicus and Sesbania rostrata have an efficiency equal to an
average fertilizer dressing (both being able to fix 80-100kg N/ha
in 45-60 days; #Becker 1995)
due to present cheap urea fertilizers the manure use is declining
at the moment in a world-wide scale. In the Sichuan basin green
manures that can be occasionally found in marginal areas are
Astragalus sinicus and Vicia sativa.
Still, legumes make up for a reasonable share of crops and they
can be nearly as effective as explicit manures. In subtropical
humid fertile environments such as the Sichuan basin around
Chengdu, legumes that are used directly as food are faba bean and
French bean (growing all over they year, main harvest in May), mung
bean, cowpea, soybean, Dolichos lablab and peanuts (harvested in
September). All of these species are relatively sturdy and due to
huge plant size also have huge nodules. For example, with Chengdu
Dolichos lablab nodules of a diameter up to 3cm can be found. The
products of nitrogen fixation go directly into the plants and their
seeds. Of course, this sometimes is responsible for the fact that
contrary to popular belief some legumes extract rather than
increase soil nitrogen content (#Rao
1981249), however adverse effects can be ameliorated if legume
stover is returned to the fields: #Toomsan (1995) reports N fixation
rates of 150-220kg N/ha for peanuts and 100-152kg N/ha for soybeans
in Thailand, and if the stover is left on the field, after
harvesting the N-rich crop some 13-100 kg N/ha are left in a peanut
field; however soybean cultivation resulted in a net loss of
37-46kg N/ha. In comparison, a crop like maize usually takes out
some 150kg N/ha.
Even in very cold environments as Murmansk (north of the Polar
Circle), where nodulation on wild Astragali and Oxytopis were
considered as unsatisfactory (#Roizin 1959), a pea crop were able to
fix 28-76 kg N/ha (#Egorov
1985 measured by ARA) and on the Qinghai-Tibet plateau (Lhasa
area between 3500-4100m altitude, ann. mean temp. 6-8oC), crop
rotation systems with barley, wheat, peas and faba beans (#Zhu 1990 #Zhou 1991) have been achieved.
In temperate grasslands (such as at Xiaman) legumes occur as
wild plants, they seldom attain the large size of crop legumes (#Zhang Xiaochuan 1989). They also
have a high protein content, but as they usually don't make up a
very large proportion of plant biomass, secondary effects such as
nitrogen conversion might be more important, this can happen by
decomposition of legumes and nodules and has been estimated at 3 to
102kg N/year*ha or 2-26% of biological nitrogen fixation for clover
and alfalfa pastures (#Ledgard
1992#Thomas 1993).
On the Qinghai-Tibet plateau, #Yang (1995) in Hongyuan also reported a
poor nodulation for high-altitude wild legumes and #Nie (1989) in Gansu achieved good
results with medic inoculation. Obviously the amount of leguminous
nitrogen fixation is also dependent on many factors, such as
temperature, soil pH and soil nutrition, salinity, soil moisture
etc. In the following some factors possibly important for Xiaman
will be discussed.
1.3.3. Temperature All leguminous host
plants so far investigated have a normal Calvin photosynthetic
cycle, with optimal temperatures of 15-25oC (#Bordeleau 1994). Most
rhizobia are thought to grow best at a temperature of 28 to 31oC
(and 28oC is indeed the standard temperature for culturing
rhizobia), but S. meliloti (medic rhizobium) grows well at 35oC,
and arctic rhizobia grow at 5-10oC (#Graham 1992). Obviously, the optimal
temperature of the symbiosis is highly dependent on the environment
it is found in: while for subtropical regions #Lie(1971) reported that a pea cultivar
Iran was nodulated at 26oC by R.leguminosarum, but not at 20oC, #Bordeleau (1994) determined
the optimal temperature for an Oxytropis maydellana - rhizobium
synthesis at 15-25oC. The Canadian research group also found that
at 10oC arctic rhizobia grew relatively faster than temperate ones,
and nitrogenase activity was detectable at up to -4oC (ibd.),
whereas in alfalfa below 8oC nitrogenase and nodulation cease (#Cralle and Heichel 1982). One
should keep in mind that nonsymbiotic plant nitrate assimilation is
inhibited by low temperatures as well (#Atkin 1994).
The evidence that temperature optima for the symbiosis are closely related to its natural origin must be taken in consideration when culturing the symbiosis in nodulation studies.
Rhizobia, especially fast-growing rhizobia (#Cao Jingle 1994A & 1994B, except R.loti and R.tropici, #Graham 1992), are more sensitive to acid than most other bacteria, which can be easily demonstrated on agar plates. In the field soil, this sometimes turns out to be a matter of practical concern, especially for S. meliloti: #Brockwell (1991) found that in Australian soils at pH=7.0 there are about 89,000 wild medic rhizobia/gram soil, but only 37/gram soil at pH=6.0. More disconcertingly, #Evans (1980) working on peas found that although legume and rhizobia do tolerate a certain level of acidity, nodulation was 10 times more susceptible to acid than soil survival of each of the partners and inoculation experiments with acid-resistant strains have been successful in the St. Petersburg area (#Fesenko 1995). At very low pH (below 5.0) excessive quantities of aluminum and manganese will be liberated below. This is not a major problem on the grassland, but might be of relevance for some areas in the Sichuan basin, where soil pH is below 5 (such as yellow soils around Chongqing with a pH 4.4-4.6, see #Feng 1996 or the Longmen Mt. SE of Chengdu). Although relatively harmless to the rhizobia, aluminum and manganese stunt plant root growth and thus influence nodulation, a problem which can be overcome by either plant cultivar selection or soil liming (Bordeleau 1994).
On the other hand, though most rhizobia which maintain a
slightly alkaline intracellular pH (#Glenn 1994) are thought to be most
efficient at neutral pH, #Tang and
Robson (1993) found a pH above 6.0 to reduce nodulation in
lupines.
The soil nutrient distribution is most intricately related to pH
levels: calcium, phosphate and molybdenum become scarce at acid pH,
and reports can be found about the stimulating influence of each of
them (for Mo, see #Tu 1992, for Ca and P, see #Graham 1992).
Legumes are more rich in potassium and phosphate than most other
plants, and interestingly phosphorus seems to accumulate in the
nodules as well as rhizobia (ibd.). So quite a lot of work has been
done to combine nitrogen fixation with P-accumulating mycorrhiza
(symbiotic fungi, #Buttery
1992#Lynd 1995).
A high content of rapidly available soil nitrogen induces some
plants to suppress nodulation (by stopping flavonoid excretion, see
#Coronado 1996). This plant
autocontrol of nodulation (#Caetano-Anolles 1991) thus averts
wasting energy when enough nitrogen can be scavenged, which can be
undesirable for the breeder or pastoralist, but if this problem
occurs it can be avoided by choosing appropriate cultivars. For the
majority of wild legumes it is still unresearched in how far they
are influenced by this.
The structure of this section basically follows the classic
books on methods by #Vincent (1970) and #Bergersen (1980); Chinese
translations of both works have been published and are widely
available. So the emphasis in this presentation is on what is new
(not mentioned in those works) or especially relevant for this
thesis.
1.4.1. Determining overall legume quantity and distribution
In natural ecosystems the first step is to ascertain whether
there are wild legumes and to get some basic data about their
biology. Host plant identification should be correct or at least
reproducible (deposit specimen in the local herbarium). Standard
methods for assessing biomass dynamics and plant communities can be
found in #Jiang 1988
An easy and objective way of sampling is along a transect, in
plant biology the most "classical" example is probably the
Californian transect experiment by #Clausen,Keck and
Hiesey(19401945,1948,1952;reviewed by #Davis and Heywood 1963) crossing two
mountain ridges (with a relative altitude difference of more than
2000m) for several hundred km, they investigated the distribution
and environmental requirements of multispecies forb genera like
Sisyrinchium (Iridaceae), Aster, Artemisia, Achillea,Horketia
(Asteraceae) and Potentilla (Rosaceae). Transects of any length
have been used to assess very diverse biological phenomena, such as
insect (#Zhou 1992) or fish
distributions (#Hong 1989).
Very recently, #Fang and Ohsawa
(1996) published data of forest distributions along the 30N
transect in East Asia (transect length: more than 1/8th of the 30N
meridian!).
Obtaining these data is laborious, but often quite low-tech, and
the more difficult problem is to analyze them appropriately and to
filter out relevant relations. As totally unknown plant
distribution data should not be assumed to be normally distributed,
for this it is recommended to employ more robust non-parametric
statistics such as Kendall's tau, the Wilcoxon-Mann-Whitney rank
tests or Chi-square tests (#Wall
1986#Sprent 1989). From
the statistical point of view, to discover a non-obvious, but not
very hidden relation (by nonparametric statistics) a number of
10-100 samples is usually optimal.
The crudest approach for evaluating nodules in the field is to
observe their color: as effective nodules contain leghemoglobin to
ensure the aereation of the nodule-enclosed rhizobia, nodule color
gives a first hint on efficiency, white being "bad" and red being
"good". Of course, these qualitive observations are not very
amenable to quantification, and so a variety of more exact methods
has evolved.
1.4.2.1. Direct approach: acetylene reduction assay
Nitrogenase is an enzyme which is not strictly specific: besides the reduction of nitrogen it also readily catalyzes the reduction of acetylene to ethylene. As both compounds are gaseous and both are not part of the natural atmosphere in significant quantities, this is amenable to gas chromatography analysis, and from the amount of ethylene measured inferences about the nitrogen fixation can be made. This has been a standard method BNF assessment during the 70s and 80s, and for example in our province #Deng (1992) used it for assessing non-leguminous tree N-fixation. However it has the serious drawback that it requires swift movement of samples to the lab (not feasible in Ruoergai) or a field-mobile gas chromatograph. Furthermore, the ARA N fixation data are just a snapshot (usual measurement times are several hours) and thus neglect that overall N fixation is in a complex homeostasis between many biotic and abiotic factors (local N content, development stages of individual plants, temperature changes etc.) and different ARA data are not always easy to compare.
1.4.2.2. Indirect approach: N labeling and depletion studies
The N content of any matter can be determined by the Kjeldahl
analysis (sulfuric acid digestion), however although legumes
usually have a higher N-content than non-legumes, absolute N
contents do not tell us much about its origin. However, different
isotopes of N allow to enrich an fertilizer with radioactively
labeled N, and to calculate the ratio of radioactive 15-N
(non-fixed) to non-radioactive 14-N (fixed) nitrogen. Soon this
technique evolved to a N depletion assay (#LaRue and Pattison 1981#Xie 1991) taking advantage of the fact
that 0.3663% of atmospheric nitrogen is actually radioactive, so
that instead of hyperradioactive fertilizer hyporadiactive could be
used; with this assay the N-fixing plants become more radioactive
than their fertilizer-fed counterparts. With increased precision of
analysis this again has evolved into an 15-N isotope fractionation
technique (#Ledgard 1992#Doughton 1992 for application
see #Bolger 1995and #Michelsen 1996); this method does
not use any fertilization, but rather directly calculates N
fixation in the field from the ratio of radioactive to
non-radioactive nitrogen.
This is conceptually very straight-forward: make a fertilization experiment and see whether there is any response in biomass production. However, N application should not be too early as otherwise nitrogen will have been lost as ammonia. Attention should be paid whether the fertilization has any adverse effects on nodulation patterns.
For long times it has been known that, when a new legume is
grown on an unknown soil, sometimes good results are achieved by
inoculating the soil with soil from different area where the crop
has been grown for a longer period of time. With the discovery of
the N fixing organism it has then become possible to apply rhizobia
directly to soils, the earliest well-known product, Nitragin, going
into production in 1896, that time consisting for pure bottled
rhizobia broth (#Smith 1992),
and subsequently a series of more sophisticated techniques evolved.
However, as it is very easy to get a microorganism into the soil
and very difficult to get it out of it, non-native strains must be
thoroughly tested for their effectiveness.
1.4.4.1. Isolation and maintenance of rhizobia
As rhizobia are concentrated in root nodules, finding rhizobia is relatively easy: just dig out a plant and smash it's cleaned nodules in a sterile environment onto on agar plate. However, this of course only finds those rhizobia that most competitively nodulate plants in the field and no latent (possible more effective, but less competitive) strains. Thus recently a variety of DNA-DNA hybridization (#Louvrier 1995) and PCR-based (#Pillai 1992) identification methods have evolved to isolate rhizobia directly form the soil. These researches have led to a recent reawakened interest in selective media, some of which are used for isolating, others for checking of rhizobia.
Unfortunately, earlier reviews (such as #Mueller 1925and #Fred 1932) weren't very optimistic on
this selective media. Moreover, some media reported in the 60s and
70s (such as #Graham 1969)
were subsequently denounced as irreproducible (#Pattison 1974) or too inhibitive
(#Bromfield 1993). Thus it
was only very recently with the advent of reliable molecular strain
identifaction methods (see section 1.4.4.2.2.) that serious
interest in agents selective for rhizobia has reawakened (since the
early 90s). Most of these reports center on media selective for a
particular "species" of the rhizobiaceae, such as S. meliloti (#Barber 1979 #Bromfield 1994#Kinkle 1994),R.leguminosarum(#Louvrier 1995), R.tropici(#Soberon-Chavez 1989),
bradyrhizobia (#Gault and
Schwinghamer 1993 #Tong and
Sadowsky 1994#Gomez 1993),
Astragalus sinicus rhizobia (#Cao
1972), Agrobacterium (#Bernaerts 1963#Schroth 1965#Clark 1969#New 1971) etc.
Due to the scatteredness of literature reports, rediscoveries
are the rule rather than the exception: for instance, a resistance
of rhizobia to copper sulfate had been reported as early as 1907
(#Simon 1907see Mueller 1925
for -negative- review), and been rediscovered by #Tong and Sadowsky in 1994 allowing for
a publication lag, probably been independently reported by #Biro in 1995and was also (then
independently) found by us with the type strain of Bradyrhizobium
japonicum. In a similar manner, a stimulating influence of 5-20 ppm
manganese on rhizobial nitrogen fixation had been reported by #Olaru and #Rocasalano in
1915(apparently independently), then been utilized for an
Agrobacterium-selective medium by #Clark (1969 inaware of the earlier
publications), and again been emphasized in paper on arctic
rhizobia metal resistance (#Appanna 1991). Interestingly, the
manganese content in #Pagan's medium (1975), a
classic medium for ex-nodulo rhizobial N fixation, is also rather
high (no explanation given). In preliminary experiments, we also
tried manganese as a selective agent, but were unable to achieve
significant results.
The most common method for medium-termed storage of rhizobia is
on agar slants, which at 4oC usually last 3-6 months. A better
method for long-term storage is in 15% glycerol at -15oC or -70oC,
but repeated freezing and thawing have to be avoided.
1.4.4.2. Identification of rhizobia
-----------------------------------------------------------------------------------------
Table T-1.4-A: Root nodule bacteria taxonomy: state of the art
(#Lindstroem 1996)
-----------------------------------------------------------------------------------------
Genus Rhizobium
R. leguminosarum biovar viciae (#Jordan 1984) "vetch rhizobia"
R. leguminosarum biovar trifolii (#Jordan 1984) "clover rhizobia"
R. leguminosarum biovar phaseoli (#Jordan 1984) "French bean
rhizobia"
R. loti (#Jordan 1984)
"lotus rhizobia"
R. huakuii (#Chen 1991)
"Astragalus sinicus rhizobia"
R. galegae (#Lindstroem
1989) "goat's rue rhizobia"
R. tianshanense (#Chen
1995proposed) "Tianshan rhizobia"
R. tropici (#Martinez-Romero
1991) "tropic rhizobia"
R. etli (#Segovia 1993)
"etl rhizobia"(French bean)
R. ciceri (#Nour 1995proposed)
"chickpea rhizobia"
R. hainanense (#Gao 1994
proposed) "Hainan rhizobia"
Genus Sinorhizobium
S. meliloti (#Jordan 1984)
"medic rhizobia"
S. fredii (#Scholla 1984)
"fast-growing soybean rhizobia"
S. saheli (#Lindstroem
1996) "Sahel rhizobia"
S. teranga (#Lindstroem
1996) "tropical tree rhizobia"
Genus Azorhizobium
A. caulinodans (#Dreyfus
1988) "stem-nodulating rhizobia"
Genus Bradyrhizobium
Bradyrhizobium japonicum (#Jordan 1984) "slow-growing soybean
rhizobia"
Bradyrhizobium elkanii (cf. #Zhang 1996) "Elkan rhizobia"
Bradyrhizobium liaoningense (#Xu
1995proposed) "very slow-growing soyb. rhizob."
Genus Agrobacterium
epiphyletic group of non-nodulating bacteria within the
Rhizobiaceae
------------------------------------------------------------------------------------------
In most soils there are many strains of rhizobia competing for each host plant, for example, #Dowling (1986) claims to have found as many as 42 different S. meliloti strains on 100 alfalfa plants on a 100m2 field with alfalfa plants! So, when working with rhizobia one cannot assume that any soil is really free of them, however indigenous strains might sometimes be of low productivity. This shows that, for doing any research on rhizobial inoculation techniques, it is very important to identify strains correctly. There are a variety of techniques available for this and present numerical taxonomy emphasizes that none of them is the ultimate way, however the information different approaches yield can vary. Recent advances in rhizobial taxonomy have been reviewed frequently (#Chen 1985, #Wang 1992, #Yang 1993, #Martinez-Romero 1994,#Lindstroem 1995).
Reviewing current methods in microbial taxonomy #Vandamme (1996), classified
techniques by their range of data generation: For very high-order
taxonomic distinctions, DNA sequencing is optimal, for a rather
broad range from the family level to species/strain differentiation
phenetic assays can be used, at the species-to-family range cell
wall structure and fatty acid analyses give good results, for
species-genus differentiation DNA-DNA hybridization and %GC content
analyses can give good results and for the strain-species
microlevel RFLP,DNA-amplification, serology and MLEE can give
optimal results. Some of these methods will be introduced
below.
The expression of the genotype is in intricate correlation with
the environment, so that phenotypic characteristics are less stable
than genetic data. On the other hand, from a practical point of
view, phenetic characteristics reveal pretty much direct
information on the organism in an environment (albeit artificial)
and the ways to culture it. So, in most studies of totally unknown
bacterial strains a phenetic characterization is still among the
first steps.
Potential host plants (selected according to the different
inoculation groups) are grown from surface-sterilized and then
rhizobium-inoculated seeds either on agar slopes or Erlenmeyer
flasks or bigger sterile or semi-sterile arrangements. After
several weeks nodulation is observed.
1.4.4.2.1.2. Biochemical tests
Biochemical tests are usually quite simple observations on
bacterial growth under certain limiting conditions, such as adverse
pH, salt concentrations, inhibiting dyes, utilization of carbon and
nitrogen resources, antibiotic resistance and more calibrated
assays such as the Gram stain, acid production, melanin production
etc.
1.4.4.2.1.2.1. Why there is no straightforward biochemical test for rhizobianess
Most bacterial species have initially been defined by their
biochemistry, and are thus - per definition - amenable for a
determination by a set of biochemical tests which categorize the
strain. Usually, by this philosophy (most successfully employed by
the Bergey's Manual of Systematic Bacteriology), it is easy to
determine at the genus and with more labor the species of a given
strain. Unfortunately, rhizobia do not fit to this definition:
there are defined as organisms to form nodules on legumes,
regardless of their biochemistry. So there are -to date- no easy
and crucial test except the host-plant assay which however often
gives false negatives.
1.4.4.2.1.2.2. Determining rhizobianess by numerical taxonomy
Due to such practical difficulties, rhizobial classification was
a very silent field in the aftermath of W.W.II, but become more
vivid again with the advent of numerical taxonomy (made feasible by
the advent of cheap computing power). Numerical taxonomy uses
similarities in phenetic or genetic data obtained by the
researchers to cluster different entities into different taxa (see
#Sneath and Sokal (1973
Chinese edition: 1984) for in-depth discussion). For example, by
the application of numerical taxonomy, #Graham (1964) showed that
Agrobacterium and Rhizobium are closely related (Rhizobium being a
paraphyletic group), this was confirmed by DNA-DNA hybridizations
(#Heberlein 1967). Of
course, to make this data reliable, regardless whether in botany
(#Stacey 1980) or
microbiology, it is recommended to apply at least 50-100
characteristics. Characters that are frequently included are carbon
and nitrogen source utilization, pH ranges, salt, antibiotics and
dye resistance, growth speed, colony morphology, growth temperature
ranges, biochemical tests etc. (#Parke and Ornston 1984#Chen 1988#Zhang et al.1991#Sun et al.1993#Gao 1994#Novikova 1994#Chen 1995).
The most common method for assessing similarity between two
strains is the simple matching coefficient which counts the ratio
of (shared positives + shared negatives) / number of all tests (#Sneath 1973), sometimes only
(shared positives) / (shared positives + differing tests) are
counted. (#Sneath 1957).
However, it must be pointed out that as we are dealing with microbe
culture growth, there is one important consideration: cultures may
vary in their growth speed and thus very similar cultures might
simply appear very different, for example:
Test 1 2 3 4 5 6
StrainA y y y n y y
StrainB n y n n n n
StrainC n n y y y y
(y=growth,n=no growth)
On a swift glance, the similarity A/C is 50%, A/B is 33% and B/C
is 17%. However, note that all differences between A and B could
also simply be ascribed to the fact that A grew better than B
(maybe the number of cells spread on the plates was higher etc.).
#Sneath (1968) has thus
developed a concept for eliminating the influences of vigor in
bacterial taxonomy which will be discussed in more detail in
section 4.
As far as the classifying algorithm is concerned, for numerical
taxonomy there are basically two approaches, one is bottom-up, the
other is top-down. When talking about phylogenetic tree the "top"
is the root (with higher taxonomic hierarchies), the "bottom" are
leaves and fine branches (with lower taxonomic hierarchies). The
bottom-up algorithm (cluster analysis) finds out which two OTUs
(operational taxonomic units, e.g. strains or clusters of strains)
are most similar and clusters them together, that is subsequently
treats both taxonomic units a single new one. This step is repeated
until all units are clustered together. By this principles the
relatedness of strains can be classified into different hierarchies
and is often represented in a tree diagram.
Methods for cluster analysis mainly vary in how to generate
hypothetical new OTUs from old ones and have been extensively
reviewed by #Sneath 1973 In
recent times, the UPGMA algorithm reviewed by #Chen (1986) is employed most widely (at
least in numerical taxonomy),i.e. if a OTU1 consists of M strains
and OTU2 consists of N strains then their Euclidian distance
(simply the square root of (1-similarity coefficient)) is the
average of the Euclidian distances of all possible comparisions
(N*M).
On the other hand, the top-down algorithm is theoretically more
recent and elegant, as it tries to find out a direct evolutionary
most parsimonious tree. It basically assumes that a tree assuming
minimal evolution is best and can proceed by setting those OTUs
most different above the roots of a binary tree. Subsequent -
always those most different - OTUs are thus clustered to the
appropriate branches of that tree (see #Zhong 1990:162 for an example).
According to this principle (always find the most different OTUs
and connect them to the most similar branch of the already existing
tree) one can generate a reasonable tree; note however that for the
mathematically challenfing ("NP-complete") problem of finding the
shortest tree most commercial software for sequence data employs
far more sophisticated optimization algorithms.
From the above-mentioned principles it can be inferred that the bottom-up approach will be especially reliable for the low-order taxa, while the Wagner-Farris top-down approach rather emphasizes higher-order taxa. The bottom-up approach also has the advantage that the effect of different ways of assessing similarity can be seen quite directly right with the clustering steps while the whole tree is needed to say anything about their usefulness in a top-down arrangement.
The classical numerical taxonomy (previous section) has a
weakness that (a) the amount of labor involved is high (b) the
choice of biochemical tests will inevitably lead to some distortion
in the taxonomy. For example, classical numerical taxonomy had
great difficulties in assigning even higher cluster. A major
breakthrough was thus #Woese's
(1987) paper who used slowly changing 16S rRNA sequences to
propose a three kingdom system (Archaebacteria,Eubacteria and
Eukaryota). We will introduce four popular approaches to obtain
data on genetic diversity, viz. isoenzyme analysis, 16 S rRNA
sequence analysis, length analysis of restriction enzyme generated
fragments (RFLP) and amplification of repetitive sequences by the
polymerase chain reaction. Again it is emphasized there is no data
from phenetic or genetic analysis methods which is a priori "best",
and a taxonomist should use data obtained by different methods(#Graham 1991).
(1) Isoenzymes
Strictly speaking, this method also use phenetic data, but at least it is close to "sequences", that is proteins from different organisms known to have identical functions were electrophoresized, stained with protein-specific dyes and the results were compared. Unless the proteins underwent different posttranscriptional modifications amino acid sequence changes should affect electrophoretic mobility (#Selander 1986). This method was first developed for eukaryotes in the 60s, and only in the 80s became widely used in microbiology, for rhizobia see (#Martinez-Romero 1991#Eardly 1990#Demezas 1991#Souza 1992).
(2) 16S rRNA sequence analysis
This name is a bit misleading: what is sequenced is not rRNA but
rather the DNA sequence coding for it, this sequence being chosen
because rRNA accounts for 80% of cellular RNA which means that it
is repeated many times. So historically, it was relatively easy to
obtain by reverse transcription. Today, choosing appropriate
primers a fragment of the desired variability can be simply
amplified by PCR, be sequenced and compared to database
sequences.
So in rhizobial taxonomy, 16s rRNA sequence analysis has been
used to for taxonomy for wider phylogenetic relationships. For
instance, #Young (1991) used
it to establish that a photosynthetic rhizobium strain BTAi lies
near the B.japonicum type strain; #Xu
(1995), #Nour (1995), #Segovia (1993) and #Martinez-Romero(1991) used it to
establish new species (Bradyrhizobium liaoningense, Rhizobium
ciceri, R. etli and R. tropici). Using 16S RNA data, #Willems and Collins (1993) and #Sawada (1993)published
dendrograms which basically has confirmed the existing taxonomic
data on Rhizobium and Agrobacterium. For an interesting example of
applying sequence diversity to one of the host plants, the New
World Astragali, see #Wojciechowski (1993).
(3) Restriction fragment length polymorphisms
In contrast to technique (2) which uses all information of a tiny fraction of the genome, techniques (3) and (4) use a fraction of information on the whole genome. In conntrast to technique (1) which analyzes mutations in exons, (3) can also detect mutations in introns around expressed exons.
The idea of restriction fragment length polymorphism is to cut
the whole genome of each organism, electrophorize it and to
hybridize it onto nitrocellulose filters containing to known
radioactively or biotin-labeled sequences (Southern blotting). This
laborious technique has been applied for rhizobia by #Kaijalainen and Lindstroem
(1989) and #Eardly
1990
(4) Genetic fingerprinting by PCR
Principles of genetic fingerprinting: since the DNA-DNA
hybridization experiments by #Britten and Kohne (1968), the
existence of repetitive DNA in eukaryotes ("C value paradox") has
been known for more than a quarter of a century. The purification
of accurate DNA replication enzymes has made DNA amplification
feasible, and the first paper introducing the polymerase chain
reaction (PCR) to the public (#Saiki 1985) dealt with the detection
of sickle cell anemia alleles in an eukaryote (man). Very soon have
repetitive sequences such as Alu (in mammalia) been used for
"genetic fingerprinting" (#Nelson
1989), i.e. the amplification of highly variably distributed
sequences to identify individuals. While 16SRNA sequence analysis
is still the standard tool for the higher-taxa bacterial
taxonomist, for rather fine intrageneric or intraspecific analyses,
genetic fingerprinting compares well with the more laborious
isozyme analysis or serological data.
There are two common approaches: as a primer either choose any
short random sequence and hope that it is adequately replicated,
this approach is called RAPD, see #Williams 1990 #Welsh and McClelland 1990 #Hui Dongwei 1992#Cao Jiashu 1995#Bingen 1995#Woodburn 1995#Fadi 1995),in rhizobium it has been
used by #Harrison (1992)
and #Dye (1995), on the host
plant medicago by #Brummer
(1995).
With an increasing amount of bacterial DNA sequences available,
more subtle repetitive elements have been found in bacteria, too
(for rhizobia see #Flores
1987), and a second (more direct) approach became possible,
that it to choose sequences which are known to be sufficiently
repeated. Actually most "arbitrary" primers used by RAPD aren't
arbitrary at all (and indeed it would be wasting time and resources
to calibrate each experiment for truly arbitrary primers). For
example, van #Belkum (1995)
terms his methods "arbitrarily primed PCR", but among others, uses
the ERIC2 (see below) primers. In other words, the distinction is
quite blurred. The most prominent repetitive elements found in the
1980s first in E.coli, then in a wide range of bacteria, were
termed REP (repetitive extragenic palindromes; #Stern 1984) and ERIC (enterobacterial
repetitive intergenic consensus) sequences. Their role is still
unclear, but except that it could be "selfish" DNA, it seems most
probable they have some control function (#Versalovic 1991), possibly
involved in DNA polymerase or DNA gyrase binding (#Dimri 1992#Lupski 1992). Versalovic (ibd.)
constructed 12 outward-directed primers (18-36bp) based on these
sequences, and used PCR and electrophoresis generate
strain-specific fingerprints. Of these primers, he chose two pairs
of primers (one pair for ERIC, one pair for REP), widely
distributed in the eubacteria, which since then have been widely
applied. Some authors (#Cassol
1994working on HIV) reported better results for REPs, others
(#Rodriguez-Barradas
1995working on the rhizobium relative Bartonella) found ERICs
yielding more information. Some only used REPs (#Georghiou 1994), others only ERIC
(#Liu 1994)
As this method is very promising for the large rhizobial genomes
(#Sobral 1991),rhizobium
workers began to use it very early (#Bruijn 1992 #Judd 1993) on S. meliloti and
Bradyrhizobium japonicum.
1.4.4.3. Assessing the effectiveness of inoculants
To date, the only reliable way of doing this is to expose plants
to rhizobia. This can be done in similar arrangements used for host
plant range determination This can be done either in the lab
(#Gibson 1980) or in the field (#Vincent 1970), e.g. by genetically
marked strains (#Wilson 1995)
allowing easy assays, for a recent review of general current
techniques see #Herridge
1995
If the productivity of a given strain has been clearly established, one can ferment it on a medium scale to produce inoculants. Although pure broth can be used for field testing, most commercial inoculants use carriers increasing the longevity of the products, such as peat (#Rodriguez-Navarro 1991), polyacrylamide (#Hedge 1992), vermiculite (#Ning 1993), soil powder (#Liu 1991) and talc powder (#Wang 1989). When producing inoculants, viable rhizobia should outpass the number of seeds by three to five magnitudes, depending on seed size (#Olsen 1994) and plant species (#Patrick 1995).
1.4.5. A word on basic research such as
physiology and genetics of rhizobia The intricate details
described in section 1.1.2.3. have been elucidated by many
physiological and genetic methods, and the rhizobiaceae are
probably among the 20 best-researched families of bacteria.
#Brockwell (1995) gives a review on the potential of engineered
rhizobia applications. Genetic engineering of rhizobia can lead to
very fast breakthroughs (such as the successful transfer of a S.
fredii 3.7kb enhancement factor to Bradyrhizobium japonicum, #Zhang 1994), but not every
apparent progress will work out that well in the field. For
example, supernodulating mutants have been created which form more
nodules than normal strains, however these supernodulating mutants
often cause more harm then benefit when applied in the field, as
they force plants to an unnatural nodulation behavior (#Buttery 1992).
Other plants have been manipulated to bear nodules by the herbicide 2,4-dinitrophenol (#Nie 1991), but at the moment it is doubtful whether the yield responses reported by these techniques (see for example #Liu 1993) have indeed been caused by rhizobial N fixation. For example, some brassicaceae are by inheritance susceptible to broad-host-range bradyrhizobia which indeed elicit nodules on these plants. However, as the brassicacean root tissue is poisonous to the bacteria, no infection occurs, and all N-fixation effects can be attributed to azospirilla accumulating outside the nodule (#Brockwell 1995).Thus, as far non-leguminous N fixation is concerned, in the moment it might be more reasonable to have a closer look at the asymbiotic root fauna to achieve direct results.
So although these are very promising approaches which are of paramount importance for our understanding of this symbiosis, other symbioses and plant pathogens (#Wilson 1995), however when embarking on research projects in this area, one should not always expect very fast breakthroughs for direct applications.
One should not forget that rhizobia only make up for half of the symbiosis, and there is good potential for legume breeding, for reviews see #Herridge 1995 (general) and #Ranalli 1995 (European pulses).
As relatively little work has been done on Qinghai-Tibet plateau
rhizobia, this thesis aimed to lay a foundation for that field:
this includes the observation of naturally occurring legumes and to
investigate their nodulation status and the soil nutrient situation
at Xiaman. A preliminary inoculation trial with vetch rhizobia was
included. It is hoped that by the identification of naturally
occurring rhizobia as well as by proposing media for identification
more directed inoculation should become possible.