Note: Descriptions are shown in the official language in which they were submitted.
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
1
METHOD OF LAND MANAGEMENT INVOLVING MICROBIAL BIOASSAY
The invention relates to a method of land management involving the
assessment of soil quality as a control parameter in practice of such method.
The intensity of ' many contemporary land-based primary production
systems (agriculture, horticulture, silviculture etc) has led to significant
ecosystem
degradation in the form of loss of soil biodiversity, nutrient depletion, soil
erosion,
secondary salinity and adverse effects on water quality. This degradation of
soils
and associated decreases in productivity and food quality; mean that
considerable research attention is being given to assessment of alternative
management of primary production systems. Strategies for alternative
management range from a more careful accounting of nutrient budgets while
still
pursuing conventional (fertilizer and pesticide based) management systems, to
'organic' management.
Numerous concerns have been raised about the likelihood that strict forms
of organic management (referred to simply as 'organic' in this application)
are
unsustainable, since outputs of nutrients may exceed inputs, with subsequent
'mining' of soil nutrients. These concerns have led segments of the industry
to
adopt an approach to sustainable land management, which integrates a focus on
organic soil amendments and zero or minimal synthetic pesticide use, with
nutrient inputs allowed from inorganic or industrially-produced nutrient
sources.
In this application, an approach based on this premise is referred to as
'integrated
land management', biological management or biological farming. Several recent
reviews have established that, in many cases, integrated land management or
biological farming improves soil quality, particularly by increasing soil
organic
matter and microbial contents, increasing beneficial microbial activity in
soils, and
improves soil physical properties. Additional wider benefits may include
reducing
pesticide residues in food crops, lower export of pollutants (particularly
nutrients
and greenhouse gases) from managed ecosystems, and lower economic inputs.
In addition, both integrated and fully organic management incur lower
production
costs associated with lower inputs of fossil fuels, manufactured fertilizers
and
agrochemicals.
An aim of integrated land management is enhancement of biological
nutrient cycling to reduce reliance on synthetic nutrient additions and to
restore
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
2
beneficial ecological function to soils degraded by conventional management.
With this in mind, considerable effort has been directed by the integrated
land
management industry towards development and application of alternative soil
amendments, or 'biostimulants' designed to facilitate or even stimulate soil
biological activity. Traditionally, such soil amendments have included:
livestock
manures; green (crop residue) manures; unprocessed mineral amendments such
as lime, rock phosphate or gypsum; or composts prepared from one or more
organic source(s). Manuring and compost application promote soil microbial
activity directly, primarily by supplying a metabolisable carbon source
together
with nutrients.
A further distinctive feature of integrated land management practices
relates to the type of soil quality data collected and the paradigms for
interpretation of such data. In attempts to reflect the underlying values of
attaining ecological balance for land under integrated land management, suites
of
soil analyses such as the Albrecht or Reams systems differ from conventional
soil
testing by including parameters specifically intended to assess holistic
ecosystem
health. However, soil chemical analyses may not provide a useful indication of
soil quality for land which is managed using integrated or organic paradigms.
It is therefore an object of the present invention to provide a method for
land management that allows the assessment of soil quality in a manner which
provides a reliable and useful indication of soil quality, particularly from
the
biological perspective.
With this object in view, the present invention provides - in one aspect - a
method for land management comprising measuring, as a measure of
effectiveness of the method, soil quality of a soil sample collected from land
under
management by a microbial bioassay including at least determining fungal:
bacterial ratio of said soil sample by substrate induced respiration combined
with
selective inhibition by multiple soluble inhibitors, wherein said multiple
soluble
inhibitors are selected with reference to the land under management; and a
nutrient application programme for a specified crop growable on the land is
controlled on the basis of the determined fungal:bacterial ratio. Such
measurement may be employed to identify the availability of nutrients such as
nitrogen, phosphorus and sulphur to plants as such availability is mediated by
soil
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
3
micro-organisms. Action to increase plant availability of nutrients may be
initiated, if required. The fungal:bacterial ratio soil quality measurement
may also
be an indicator of sustainability of the land management method.
The soil quality measurement step or aspect of the method reproducibly
generates microbial biomass data and fungal:bacterial ratio data having an
inhibitor additivity ratio (IAR) of 1.00 ( 0.15) where:
IAR = (A-B)+(A-C)/(A-D)
and A is total soil biomass, (A-B) is the difference between total soil
biomass and biomass in the presence of fungal inhibitor(s), (A-C) is
difference
between total soil biomass and biomass in the presence of bacterial
inhibitor(s)
and (A-D) is the difference between the total soil biomass and biomass in the
presence of both fungal and bacterial inhibitors at similar concentrations to
A and
B. The inhibitors employed are selected or calibrated with reference to soil
type
and indigenous microflora of that soil type; and/or soil texture in order to
avoid
inhibition of non-target bacteria or fungi, problematic in prior art methods.
Therefore, prior calibration to assess a suitable pair may be required.
Calibration
involves selecting the best inhibitor pair that shows maximum respiration
inhibition on a wide range of soil types; and determining the best inhibitor
concentrations that yield the target IAR of 1.00 ( 0.15). Such IAR indicates
that
the inhibitors have acted only upon the target organisms such that measured
fungal:bacterial ratio has acceptable reliability. An IAR of greater than 1:00
(
0.15) signifies non-target, and less than 1.00 ( 0.15) inadequate inhibition.
Fungal inhibitors, such as cycloheximide, and bacterial inhibitors, such as
kanamycin, as selected by the Applicants for tests on a Western Australian
soil at
the University of Western Australia, are applied to soil samples in multiple
different concentrations. Concentrations may be adapted to different suites of
commonly occurring soil microorganisms such as heterotrophic bacteria and
fungi, such as arbuscular mycorrhizal fungi, fluorescent pseudomonads and
actinomycetes and according to the suites of microbes actually inhabiting the
land
under management. Microbe nature varies with soil type and soil
characteristics.
A total of 7 soil treatments to establish inhibitor efficiency has been found
beneficial. The inhibitors are applied to the soil sample, in accordance with
the
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
4
method, in solution form. Talc or similar substrates are advantageously
avoided
as it is not necessary when inhibitors are applied in solution form.
Glucose or similar carbon source or respiratory substrate for microbes is
added to each soil sample during the analysis. The carbon source may be added
following addition of inhibitor.
Moisture in the sample is controlled to ensure proper dispersion of inhibitor
and glucose and aerobic conditions.
The measurement of fungal:bacterial ratio may support or supplement
measures of total microbial biomass as a step in assessing soil biological
activity
as part of the land management method.
Soil quality data obtained in accordance with the land management
method, which may -for example - be conducted in accordance with Australian
Patent Application No. 57966/01, the contents of which are hereby incorporated
by reference, form a further aspect of the invention which may be compared
with
objective standards for the soil in the managed land and a nutrient
application
programme maintained or varied to reduce any adverse variations from these
objective standards. Reliable soil quality data assists this. Such nutrient
application programme is preferably a biologically acceptable nutrient
appiication
programme.
In such embodiment, the present invention provides a biological method of
managing crop production comprising the following steps:
a) selecting an area to be farmed with a specified crop;
b) evaluating said area, which evaluation includes conducting physical
and/or chemical analysis of the soil environment of said area;
c) determining objective standards for soil and crops in said area and
a biological nutrient application programme for the area aimed at achieving
those
of the objective standards which are specific to the specified crop, the
programme
consisting of application of biologically acceptable components not known to
cause genetic modification of plants; 0
d) conducting the biological nutrient application programme;
e) conducting testing of soil and specified crops in the area for
measuring compliance with said objective standards; and
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
f) adjusting the biological nutrient application programme, as
necessary, to reduce adverse variations from said objective standards and
wherein the biological nutrient application programme is conducted to
stimulate
microbial activity to promote plant availability of nitrogen and in response
to
5 microbial bioassay data, most advantageously fungal:bacterial ratio data for
soil
samples from the area to be farmed with the specified crop. This ratio is
determined by a method, as described above, in which the multiple soluble
inhibitors are selected with reference to the area to be farmed with the
specified
crop.
Nutrients should, more desirably must, be introduced to the soil only in
biologically acceptable form, the objective being to implement a nutrient
application programme, controlled to increase the biological activity of soil
in the
land while maintaining ecological sustainability. Organic form may be
acceptable
in certain circumstances. Thus, phosphorus may be introduced in the form of
phosphate rock and nitrogen may be introduced through use of animal waste
products and fish based products. Nitrogen control, in terms of controlling
nitrogen input to land under management, is important. While nitrogen is
essential to crop growth, excess nitrogen can create problems by increased
susceptibility of crop to disease and insect attack. Nutritional content may
also be
reduced. These issues may be addressed by growing of plants (such as clover
and legumes) and enhancement of nitrogen fixing microbes, enhancing nitrogen
fixation from the atmosphere. Nutrients may also, and advantageously, be
supplied by biological fertilizer products such as that available under the
trade
mark ERAPHOS, subject of Australian Patent Application No. 18362/01, the
contents of which are hereby incorporated by reference.
The land management method, as described in Australian Patent
Application No. 57966/01 is an integrated land management method concerned
with ecological sustainability which may be applied to the growing of various
crops such as wheat and olives being examples of specified crops. The method
may also be used to assess potential for various crops to be grown in the land
under management. For example, data generated by the method may assist in
identifying crops suitable for growing in the land under management.
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
6
Such cash crops may be grown in an initially nitrogen deficient soil, thus
warranting a strategy to improve biological activity and better plant
availability of
nitrogen through biological mineralization of nitrogen. Soil bioassay to
assess
total microbial biomass and the fungal:bacterial ratio is included,
advantageously
as above described, to assess the efficacy of the method to improve biological
activity and better plant availability of nitrogen and organic matter through
sufficient humification of the soil. Using such data, a biological nutrient
application programme may be controlled to achieve these objectives. This
forms
a further aspect of the present invention. In the case of olives, a preferred
range
of fungal:bacterial ratio is 2:1 to 5:1 so, if a soil is fungally deficient,
the method
will include steps, such as administration of fungal inocula and fungal
enriched
compost to increase the ratio towards the preferred range. Conversely, if a
bacterial deficiency is identified a strategy to augment soil biological
activity may
be implemented. Microbial inocula and bacteria rich fertilizers such as
compost
tea or fish emulsion, may be applied to increase microbial activity.
An additional step of mycorrhizal bioassay, such as by measuring
mycorrhizal inoculum potential, may be included within the method or as an
additional measurement of soil quality. In particular, the presence of
mycorrhizal
fungi is an indicator of the balance of soils and the sustainability of the
land
management method. Changes in mycorrhizal inoculum potential may also be
tracked, with fungal: bacterial ratio, in response to changes in land
management
techniques. The nutrient application programme is then controlled as a
function
of fungal:bacterial ratio and mycorrhizal inoculum potential.
Mycorrhizal inoculum potential may be measured in the field, rather than
through the agency of laboratory testing. The plant roots are cleared and
stained
as described, for example, Koske, RE, Gemma, JN (1989) A Modified Procedure
for Staining Roots to detect V-A Mycorrhizas, Mycological Research 92: 486-
488,
Phillips, JM, Hayman, DJ (1970) Improved Procedures for Clearing Roots and
Staining Parasitic and Vesicular Arbuscular Mycorrhizal Fungi for Rapid
Assessment of Infection, Transactions of the British Mycological Society 55:
159-
161, the contents of which are hereby incorporated by reference. Stained roots
are laid down on microscopic slides or gridded Petri dishes and mycorrhizal
colonization (inoculum potential) scored under a microscope.
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
7
Simple equipment, avoiding expensive laboratory equipment such as
autoclaves - for example by using electric pressure cookers, allows
examination
of the mycorrhizal status of any crop species in its native environment and in
its
active growth phase. This information can be used for growth model
construction
and simulation. By confining the tests to the field, time may be saved in
sample
handling and processing. The direct testing of crop roots is advantageous over
collecting paddock soils and performing mycorrhizal bioassays in glasshouse-
grown plants, as the plant and the environmental conditions are totally
different in
glasshouse bioassays.
The practice of the method of the invention may be more fully understood
from the following examples of application of methods of the invention. The
description is illustrative of the methods rather than limiting and its
application to
wheat farming and olive growing situations is demonstrative of only two types
of
crop to which the method could be directed. Other crops, such as palm fruit to
be
used in the production of palm oil, may also be managed in accordance with the
method of the invention.
Examples
Example 1
Soil samples were obtained from a wheat paddock. Measurement of soil
quality was conducted in accordance with the following protocol in accordance
with the method of the invention.
1. Using 30 ml McCartney bottles, prepare the following experimental
trials in duplicates or triplicates:
There are seven soil sample treatments, this number being beneficial in
the practice of the method.
Table 1. Soil sample treatments.
Column 1 shows 2 g portions of the soil sample in 8 separate tubes.
Column 2 shows the treatment each tube receives. The moisture content
of the sample in each tube at this stage is about 15 % water holding capacity.
Tube Treatment
name
A None
B Cycloheximide (fungal inhibitor); Rate, 2 mg inhibitor/tube
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
8
C Kanamycin (bacterial inhibitor); Rate, 3 mg inhibitor/tube
D Cycloheximide and Kanamycin mixed together at rates
shown in columns B & C
E Cycloheximide; Rate, 3 mg inhibitor/tube
F Kanamycin; Rate, 2 mg inhibitor/tube
G Cycloheximide and Kanamycin mixed together at rates
shown in columns E & F -
H Cycloheximide and Kanamycin mixed together at rates
shown in columns B & F
The inhibitors are mixed with water to form solutions of inhibitors, absent
talc or other substrate, and applied at rates of 200,uUtube.
2. Incubate for 1 hr at room temperature
3. Add glucose at rates of 50 ,uUtube (8 % solution, w/v) and seal the
bottles with rubber septa.
5. Incubate at 25 C for 4 hrs in the dark
6. Calibrate the IR gas analyzer using 0.1 ml, 0.2 ml and 0.3 ml 4.95 %
ultra pure standard CO2 gas
7. Syringe out 1 ml portions of the headspace gas from the sample
bottles and inject into the calibrated gas analyser
8. Measure the peak heights of standards and samples
9. Calculate the biomass for each sample using the following equation
of Anderson, J and Domsch, K "Measurement of Bacterial and Fungal
Contributions to Respiration of Selected Agricultural and Forrest Soils", Can
J
Microbiol, Vol 21, 1975, p314.
Pg C02-C g"' soil =((Asample x 10000/Bstd)/103 xV X K))-((Ab-ank X
10000/Bstd)/103 x V x K))
where:
Asampie = Peak height (mm) of sample
Abiaõk = Peak height (mm) of blank bottle
BStd = Peak height (mm) of standard gas (1 % C02)
V = Head space volume of bottle
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
9
K = Conversion constant for pl to pg of C02-C (assuming 1
atmosphere pressure: 1.7995 pg CO2 = 1 pl C02 at 25 C and 1 atmosphere =
0.4908 pg C02-C)
10. Calculate the parameters, and pick up the most acceptable fungal
bacterial ratio, i.e. the one with an IAR closest to 1.00, from one of the
following 3
datasets:
Table 2. Datasets generated from the incubation trials.
Dataset Treatment* Fungal Bacterial Fungal Inhibitor
biomass biomass bacterial additivity ratio
ratio (IAR)
1 A B C D (A-B) (A-C) (A-B)/(A-C) (A-B) + (A-C)/(A-
D)
2 A E F G (A-E) (A-F) (A-E)/(A-F) (A-E) + (A-F)/(A-
G)
3 A B F H (A-B) (A-F) (A-B)/(A-F) (A-B) + (A-F)/(A-
H)
*Letters A through to H correspond to tube names in Table 1.
Table 3. Example of an actual dataset from a paddock soil sample.
Dataset Total Fungal Bacterial Fungal IAR
microbial biomass biomass bacterial
biomass ratio
266A 25.6 8.9 3.5 2.5 1.11
266B 25.6 10.1 4.7 2.1 1.19
266C 25.6 8.9 4.7 1.9 1.28
Biomass values are in pg/g dry soil
It is clear from Table 3 that dataset A has yielded an IAR 1.11, within the
acceptable range, and hence the biomass and fungal bacterial ratio of this
dataset are the most acceptable numbers and may be used in the control of an
integrated land management method.
Example 2
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
Olive trees were planted in order to establish an olive grove. The grove
lacked legume species, thus reducing photosynthetic carbon root exudates that
may act as food source for different species of microorganisms. Accordingly,
action was proposed to enhance growth of diverse micro-organisms in order to
5 optimise plant growth. Specifically, optimal fungal:bacterial ratios for
olive growth
lie in the range 2:1 to 5:1.
Fungal:bacterial ratio measurements made by a method as above
described, confirmed plant tissue testing showing low levels of nitrogen, as
an
indicator of insufficient microbial activity to mineralise enough nitrogen to
sustain
10 crop growth. In addition, the organic matter in the soil of the grove was
insufficiently humified. One soil sample had a fungal:bacterial ratio of 1.2:1
and
other soil samples had lower fungal content and indicating a potential fungal
deficiency. The range for microbial biomass across the samples was 26.1-45.1
pg/g and that for mycorrhizal inoculum potential was 29 to 46%.
In order to address these issues, fertilisation strategy was altered. A
fungal dominant or fungus inoculated compost blend was applied, as a fungal
augmentation strategy for adding sufficient quantity of organic matter to the
region of soil around each olive tree to sustain growth for a considerable
period.
Mulching with woodchips supplemented the organic matter. Such mulch is
primarily composed of cellulose and lignin. Organisms well adapted to
decompose such material are saprophytic fungal species with which the compost
was enriched. On decomposition, the product simple sugars and carbohydrates
act as food source for actinomycetes and bacterial species. Predation from
grazing organisms such as nematodes and protozoa (which mainly consume
bacteria) will cause release of - among other compounds - organic nitrogen to
be
taken up by the olive roots. Accordingly, the total biomass of fungi and
bacteria in
the soil is the main source of plant available nitrogen.
Accordingly, promotion of such nitrogen cycling and mineralisation
mechanisms was the objective for management of the olive grove. Integration of
microbial inoculums such as compost teas, fungal, bacterial and protozoa
infusions was therefore employed in the nutritional program to boost
biological
activity and plant availability of nitrogen. Compost tea and fish hydrolysate
were
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
11
applied in combination. Compost tea introduced a diverse range of beneficial
bacterial, fungi and protozoa species in high population.
Specifically, an annual nutrient applicant programme involved the following
stages:
Stage 1
Fertigation initiated of blended molasses, soluble kelp powder (sold under
Seagold trade mark) and fish hydrolysate. Such products could be applied
mixed, separately or sequentially. The administration rates of these products
were:
Molasses 3-5 UHa
Kelp Powder 300g/Ha
Fish Hydrolysate 5-10 UHa
Stage 2
A first application of fungal dominant compost tea occurred within 3-5 days
of applying the molasses/kelp powder/fish hydrolysate blend in Stage 1. Such
application was as a soil drench to the base of each tree at a rate of 50-100
UHa.
Addition of a commercial Azotobacter inoculum to the compost tea introduced
nitrogen fixing bacteria to the soil system.
Post Compost Tea (CT)
After applying the above CT and subsequent applications, a follow up food
source application was made within 3-5 days. This was selected to be the same
as the above blend at the same rates to provide continual food sources for the
organisms.
Protozoa or hay Infusions
An organic source of hay was used for this preparation.. The root crown
and stalk of hay are loaded with protozoa. As the hay dries down the protozoa
hibernate until moisture levels rise. A simple preparation was made by
infusing a
small amount of hay into tepid water and leaving for 5-7 days. Protozoa
consume
bacteria and produce ammonium as a waste product which is rapidly converted
into nitrate (nitrogen mineralization). Such a preparation was applied after
the first
CT application to enhance protozoa activity.
CA 02601042 2007-09-12
WO 2006/096926 PCT/AU2006/000352
12
Remainder of growing season
Maintenance fertigations of CT at a rate of 50UHa at monthly intervals
alternating with food source applications and protozoa infusions if nitrogen
stress
is still present. Advantageously, at least three of these maintenance
applications
occur after the initial inoculation. Foliar or trichoderma sprays may be mixed
with
CT application. The efficacy of trichoderma as a biocontrol of plant pathogens
may be enhanced if used in combination with compost tea.
As growth progresses, determined fungal:bacterial ratios are expected to
increase towards the target fungal:bacterial ratio range of 2:1 to 5:1. Plant
tissue
analysis for nitrogen is expected to confirm increasing nitrogen availability
to the
olive trees.
Modifications and variations to the method of the present invention may be
apparent to the skilled reader of this disclosure. Such modifications and
variations are within the scope of the present invention.
