Hawai‘i Native Plant Microbiome Manual
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Transcript of Hawai‘i Native Plant Microbiome Manual
written by:
Richard L. Quinn, ASLA
(updated June 2015)
ECOSYSTEM RESTORATION SERIESHAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PRODUCED BY:
NOTE:
This manual has been produced as part of a series on Hawaiian native ecosystem restoration.
PRODUCED BY: HHF Planners
address: HHF Planners 733 Bishop Street, suite 2590 Honolulu, Hawaii 96813
phone: 1-808-545-2055
web: www.hhf.com
Originally published in Oct 2014.
Graphics: All graphics and photos are the property of HHF Planners Inc.
TABLE OF CONTENTS
Introduction 1
Understanding Mycorrhizae 2
Essential Forest Fungi 3
Benefi ts of Mycorrhizal Fungus 4
Key Concepts 4
Important Terms 5
Invasive Plants Change Native Soils 6
Don’t Do / What to Do 7
Soil Microbes in Native Forests vs. Non-Native 8
Inoculum 9
Understanding Endophytic Fungi 11
Spreading Endophytic Fungi 11
Rhyzobacteria and other Soil Bacteria 12
PLant Viruses 12
Soil Nutrients 12
Why Phosporus and Soil pH Maters 12
Soil Amendments 13
References 15
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 1
HAWAI‘I PLANT MICROBIOME - INTRODUCTIONMuch like the stomachs and skin of animals harbor a rich array of benefi cial bacteria
needed for digestion and immune response, plants harbor essential microbial
networks that act as an extension of the plant’s biology and physiology. A single
plant can be considered as a complex ecological community in and of itself.
The plant microbiome includes a complex symbiotic interaction and association of a
plant with bacteria and fungi, both within plants cells, between the cells, and on the
surface of plant leaves, stems and roots. The composition of the plant microbiome
can have an important infl uence on the success of a plant. Just in the last few years,
advances in DNA sequencing technology have opened up an exciting new frontier
of research that will have a profound impact on approaches to agriculture as well as
native ecosystem restoration.
The microbiome of a native plant in its native ecosystem is signifi cantly diff erent
then the same species of plant grown in nursery conditions and out-planted in an
urban setting. It is becoming apparent that the plant’s microbiome is the key to the
plant’s health and productivity. By recognizing how plants are colonized by various
bacteria and fungi, and how the native microbiome can be supported in ecosystem
restoration eff orts, greater success can be achieved with a higher level of protection
of earth’s biodiversity.
ESSENTIAL SOIL ECOLOGY FOR NATIVE FOREST RESTORATION
2 PLANT MICROBIOME HHF Planners - Updated June 2015
Understanding the ecology of native soils is essential to successful restoration of
native forest biota. Recent research has revealed complex associations between
the health of the soil rhizosphere (the thin top layer) and the health and survival of
endemic native plants.
UNDERSTANDING MYCORRHIZAE Mycorrhizae are fundamental to ecosystem function. They are a complex association
between fungus and plants that can work to the benefi t of both, and are an essential
component to the native Hawaii ecosystem. There are basically two important
kinds of mycorrhizae of concern to the native ecosystem, Endomycorrhizae and
Ectomycorrhizae.
The majority of tropical hardwoods and ferns, including a majority of native Hawaiian
endemic plants, form important endomycorrhizal associations. One simple fact of
the ecosystems of the Hawaiian Islands that is of paramount importance to native
plant restoration is the understanding that mycorrhizae, NOT ROOTS, are the main
purveyors of nutrient and moisture uptake by most all endemic native Hawaiian
plants in the wild. In addition to nutrient uptake, the association of mycorrhizal fungi
to host plants essentially acts as the IMMUNE SYSTEM of the native plant. To a great
extent, to plant native Hawaiian plants without recognition of the soil ecology that
they evolved with, as often happens when planting them in urban or heavily invaded
soils, is to condemn them to failure, and create results that will be disappointing,
frustrating, and wasteful of resources.
Native mycorrhizal fungi are abundant in undisturbed native forests, but are
destroyed by soil disturbances and invasive plants. They are always missing from
newly graded sites. The lack of mycorrhizal fungi on disturbed sites can require
inoculation treatment to create a mycelium network colonization needed for a
healthy functioning ecosystem. Mycorrhizal fungus have a mutualistic relationship
with plant roots. Mycorrhizal fungi act as a physical extension of a plants root
system. They can link the roots of diff erent species of plants together, forming a
larger organism that shares resources. They are essential to restoration. When native
plants are grown away from their native soil ecology, and outplanted in urban areas
or highly disturbed sites (such as sites with mostly non-native vegetation), they
are essentially without a immune system they have evolved to need. They are less
able to compete against weeds, insect pests, and diseases. For example, research
indicates that mycorrhizal fungus can help protect a host plant from fusarium wilt,
phytothera, and nematodes pathogens, common enemies of native plants such as
Koa and Ohi‘a.
The evolution of native Hawaiian plants was under conditions of poor soil nutrition
and slow nutrient cycling, which encouraged a dependence on mycorrhizal fungus
and bacteria for the synthesis and uptake of phosphorus (P), nitrogen (N), and other
nutrients. Disturbance by excessive forest clearing, cattle grazing, fi res, and other
disturbances, can reduce the balance of native mycorrhizae, and the conditions then
become ideal for succession by non-native invasive plants. Many invasive plants,
such as Albizia trees, have a quicker nutrient cycling which can further shift the soil
biota away from supporting the recovery of native plants.
4 PLANT MICROBIOME HHF Planners - Updated June 2015
Soil Restoration should be a FUNDAMENTAL Consideration of Ecosystem RestorationThe most important type of mycorrhizal fungus for native
Hawaiian plants is called Arbuscular Mycorrhizae, or AM
for short, a type of endomycorrhizae. Urban conditions
greatly reduce the colonization of AM fungi. Disturbed soils,
eroded soils, or soils with predominantly highly invasive
species greatly reduces AM fungi. Soils that have been
subject to extensive use of fertilizers, pesticides, will have
low AM fungi. This gives native plants a great disadvantage
when planted in urban soils.
BENEFITS OF MYCORRHIZAL FUNGUS• Improved drought tolerance. Increased water uptake.
• Enhanced nutrient uptake.
• Enhanced leaf chlorophyll levels
• Resistance to disease pathogens, such as fusarium wilt
and Phytophthora, Pythium. Increased resistance to root
feeding nematodes. 17
• Enhances the establishment of benefi cial soil bacteria,
including nitrogen fi xation bacteria.
• Enhances tolerance to salt stress, soil acidity, and heavy
metal toxicity (may be indirect eff ect of improved P
nutrition).
• Reductions in noxious invasive weeds
• Soil building , erosion control, water absorption
• Plant growth hormones - AM increased production
of plant growth hormones such as cytokinins and
gibberelins;
• Helps to Link plants together into a “super-organism” that
enables the sharing of resources.
• Improves self-seedling establishment and regeneration of
native plants. 27
• Reduces or eliminates the need for artifi cial fertilizers and
pesticides.
The combination of benefi ts that mycorrhizae confer
allow plants to thrive with smaller root volume then non-
mycorrhizal plants, thus allow for greater diversity and
density within the same forest space. A greater diversity
and density of plant species supports a greater number of
pollinators, to the benefi t of individual species.
Mycorrhizal plants take up more phosphorus and grow
faster (or accomplishing the same growth rate, but with
a smaller root mass) than non-mycorrhizal plants in
nutrient poor soils. In landscape or restoration projects,
low phosphorus levels could easily be mitigated with the
addition of fertilizer, but that would negate the many other
critical benefi ts that native plants obtain from AM fungi,
such as an enhanced immune system and enhanced soil
structure.
Interestingly, well established AM fungi (AMF) have been
shown to prevent the invasion of non-mycorrhizal plants
(such as Brassaia). However, once soil is disturbed or forest
composition is changed through over clearing or grazing,
Key Concepts
• Mycorrhizae enable native plants to thrive in poor soils and marginal rainfall.
• In addition to providing nutrients, Mycorrhizae also provide an immune system to the host plant against plant pathogens.
• Mycorrhizae can also stimulate important plant hormones and plant defenses.
• Mycelium of mycorrhizae fungus binds together a forest ecosystem to share resources as a larger single “organism”.
• Mycorrhizae activity helps to bind soil particles together, improving soil structure for the benefi t of plants.
• Mycorrhizae can enhance the success of seedlings under the canopy of mother plants with low light and nutrient levels, by inter-connecting seedling roots to the mother plant roots. 18
• Mycorrhizae colonization can help plants to capture and store suffi cient reserves to facilitate mast fruiting (periodic heavy fruiting). 18
• Mycorrhizae are destroyed in disturbed and invaded forests.
• Allelopathic chemicals produced by invasive plants can destroy native mycorrhizae, shifting the soil ecosystem to the benefi t of the invasives.
• Native mycorrhizae are essential to a healthy native Hawaiian ecosystem.
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 5
invasive plants can change mycorrhizal colonization of soils
to the detriment of the re-establishment of native plants.
Recent research has shown that allelochemicals (phytotoxic
substances) produced by invasive plant roots and leaf litter,
such as that from Eucalyptus and Casuarina trees, can be
persistent in soil even after tree removal, and might limit
the re-establishment of mycorrhizal populations as well as
native plant seedling germination.
The external hyphae of some AMF fungi may spread 10-12
cm (4 inches) from the root surface, eff ectively multiplying
the surface area of the actual roots by 100 times. The smaller
diameter of AM fungi hyphae can explore micropores
in the soil that actual roots cannot reach. In addition to
mechanical access of nutrients, AMF may have chemical
mechanisms, such as acidifi cation of the rhizosphere and
excretion of chelating agents.
Mycorrhizal fungi can play a vital role in the formation and
enhancement of soils and soil structure. Resent research
Important Terms• Ectomycorrhizal (EMF): Fungus does not enter root cells, but weaves mycelium on fi ne root surfaces and between root
cells. Some produce mushrooms. Only a few endemic Hawaiian plants are considered to be Ectomycorrhizal. In general, ectomycorrhizal fungus play an important role in temperate zone forests.
• Endomycorrhizal: Fungus enters root cells. Incudes arbuscular (AM) type of mycorrhiza. Most native Hawaiian plants are endomycorrhizal. Do not produce mushrooms.
• Endophytic Fungi: Inhabit above ground tissue in plant leafs and stems. May benefi t plants, such as increasing insect resistance. They are found in all species of land plants. Understanding the ecological and evolutionary signifi cance of foliar endophytic fungi may be critical to preserving some species of endangered native Hawaiian plants.
• Arbuscular Mycorrhizae (AMF) (AM): A type of Endomycorrhizae that is more abundant in tropical soil than in temperate regions. Forms arbuscules (tree like appearance) in plant cells. Also extensive mycelium in the soil, but not in an organized fashion. Not so host specifi c, but have a large range of hosts. The most common type of mycorrhizae in nature. Has large spores, just visible to the naked eye, round yellow-orange structures.
AM Fungi are of extreme importance for plants growing in nutrient-defi cient substrates such as in volcanic and sand dune environments.
• Rhizobial Bacteria: Soil bacteria that helps plants, such as Koa, Ohai, and some other native plants to fi x nitrogen. Rhizobial bacteria work in harmony with mycorrhiza for the benefi t of the host plant. The type of AM mycorrhizal colonization can determine the diversity and abundance of the bacterial community.
• Rhizosphere Ecology: The rhizosphere is the zone of soil in the immediate vicinity of a root system. This is the zone of action for soil bacteria and fungi interaction with plant roots. The rhizosphere can have a signifi cantly different pH and soil nutrient profi le than deeper soil.
• Mycelium: The mycelium is the branching, root-like hyphae threads extending from fungus that absorbs nutrients from soil and bind together the rhizosphere for the symbiotic benefi t of plants and micro-organisms.
• Saprotrophic Mushrooms: Common mushrooms that live off dead organic matter. These mushrooms are not mycorrhizal. Shiitake and button mushrooms.
• Ectomycorrhizal Mushrooms: These are types of ectomycorrhizal fungus that produce visible fruiting bodies. These mushrooms include matsutake, truffl es, morels, and porcini mushrooms. (Tuber spp. Boletus spp. Cantharellus spp., Lactarius spp., Leccinum spp., spp.)
• Mycorrhizal Fungus: The Mycorrhizal fungi can protect the roots from disease organisms, through simple spatial interference, by improving nutrient uptake, and by producing glomulin and other metabolites that inhibit disease. Plant stress can be reduced because mycorrhizal fungi can solubilize mineral nutrients into forms available for translocation to the root system in exchange for sugars provided by the plant. Fungal hyphae, and the bacteria they encourage, are the primary agents that bind soil particles into soil aggregates. Since mycorrhizae confer different benefi ts to different species, in a subtle but important way, it is the soil mycorrhizae that ultimately help to direct the composition of the native forest, and the balance and diversity of species within.
• Plant MicroBiome: The interaction and interdependence between plant species and essential micro-organisms, such as Rhizobial bacteria, mycorrhizal fungi, and endophytes. The plant microbiome includes the Rhizosphere area around plant roots, inside and in-between plant cells, and on the surface of leaves and stems.
6 PLANT MICROBIOME HHF Planners - Updated June 2015
also indicates that mycorrhizae play an important role in
the sequestration of carbon in the soil. Research has shown
that native Hawaiian soils might have a high potential
at sequestering carbon, based on the active production
of a carbon capturing substance called Glomalin, which
mycorrhizal fungus produce. 22
Mycorrhizal colonization is lower in very dry, very wet,
or nutrient rich soils. When soil moisture and fertility are
increased past optimal levels, the dependence of plants
on mycorrhizal symbiosis decreases to a point such that
the plant becomes immune to colonization. Also, soil type
and pH can aff ect colonization success, as well as host
plant species type. Although most any mycorrhizal species
can partner with most any plant, is seems that many plant
species can selectively choose specifi c mycorrhizal partners
that are of the greatest benefi t. They apparently do this
through complex chemical cues that help guide specifi c
mycorrhizae hyphae over other mycorrhizae species to the
plant root for infection.
The inoculation of plants with AM fungus is complicated
by the fact that the fungus cannot be grown without the
use of a host plant, which limits the practical production of
inoculation material in large quantities.
Research has shown that a reasonable goal for the
successful colonization of native plants is that over 40%
of the roots of a plant have the presence of mycorrhizal
fungus.
Applying AM fungi inoculation to a small central portion, or
“isalnd” ,of a native landscape or restoration project might
be a practical way to enable a wider inoculation by natural
processes, as presumably once established, fungal spores
will travel to surrounding areas that have native plants and
are receptive to those fungal species
Studies indicate that the inoculation of soil with
mycorrhizae can be useful in restoration eff orts. “The results
of the present study and other studies on the mycorrhizal status
and growth responses of Hawaiian plants to inoculation with
AMF support the hypotheses that endemic Hawaiian species
evolved in P-limited soils and that the majority of the nearly
1000 extant species are derived from founder species with high
EMDs. As a consequence, it appears essential when restoring or
augmenting plant communities to ensure that endemic species
are inoculated before or during outplanting to native soils that
are lacking in AMF or have low mycorrhizal inoculum potential.
Such soils are likely to be found in disturbed or barren sites, the
very sites likely to require restoration.” 8 - J.N. Gemma, et al
AM fungi are greatly reduced or eliminated from
commercial nursery plant production, due to the use of soil-
free media, high rates of fertilizer and fungicide applications,
and propagation from clonal cuttings from limited (usually
non-wild) plant sources.
Some nursery grown plants and newly outplanted seedlings
do have some mycorrhizal colonization, even with the
attempts at sterile growing conditions. “The high incidence
of mycorrhizae in non-inoculated rooted cuttings suggested
that at least some species carry propagules of the VAM fungi on
the surface of their stems. Splashing rain and blowing soil may
deposit the fungi on the stems”. 9 - R.E. Koske and J.N. Gemma
INVASIVE PLANTS CHANGE NATIVE SOILSInvasive species tend to produce a greater mass of leaf litter
that decomposes more quickly than that of native plants,
which can shift soil pH, nitrogen, phosphorus, and other
soil nutrients, to the detriment of the native ecosystem. 15
Figure 1: Mycorrhizal fungus can appear as fi ne white “roots” in upper layer of soil.
Figure 2: Fungus mycelium on decomposing leaf.
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 7
DON’T DO THIS!• Don’t Fertilize after outplanting! Fertilization with N and P suppresses mycorrhizae, can destroy important soil bacteria, and
enhances weed growth. If needed, use only a low-Phosphorus fertilizer. Foliar iron, Zinc, manganese, and magnesium might be of benefi t to native plant restoration, particularly on sites with high pH and high phosphorus. Fertilization in nursery setting might be unavoidable, but discontinue the use of high N and P fertilizer near time of outplanting.
• Don’t Over Water! Over watering enhances weed growth.
• Don’t Till the soil! Breaks up the hyphal network.
• Don’t compact soil! Compaction breaks up the hyphal network.
• Don’t Remove leaf litter, branch litter, logs!: Slow decomposing leaf litter (such as from most native plants) can host important endophytic fungus, and can act as mulch.
• Minimize the use of pesticides and fungicides! These can kill or reduce mycorrhizal fungus. Some use in nursery setting might be unavoidable, but minimize use after out-planting.
• Nursery potting soil should be less than 20% peat.: Excessive peat moss reduces colonization of mycorrhizal fungus.
• Using soil as an inoculum (taking native soil and using it to inoculate AMF) is an ineffi cient and marginally effective means of transferring AMF, and can transmit unwanted pathogens. The use of soil as inoculum should be only as a last resort. 10
• Don’t use off-the-shelf mycorrhizal inoculants from mainland companies. Unlikely to be the best strain of fungus, and will introduce non-native fungus species that might displace important native AMF. Also, maybe quality control and shelf life issues. Introduced AMF might be more benefi cial to invasive plants than to native plants. My be illegal in Hawaii without proper import permit.
WHAT TO DO• Add Humic Acid: Humic acid has been shown to enhance the activity of mycorrhiza. A typical application might be 2 to 4
pounds of dry product, or 1 to 2 gallons of liquid product, per acre.
• Add wood mulch: preferably coarse mulch from hardwood sources.
• Add fresh cut logs: Logs of all sizes placed on soil surface parallel to slope, can help retain leaf litter on soil surfaces, can host fungus, reduce weeds, and retain soil moisture.
• Add native mycorrhizae to nursery plants: at least 4 weeks prior to outplanting.
• Remove aggressive non-native plants that have a high rate of nutrient recycling and/or low mycorrhizae associations.
• Hope for soil that is low in phosphorus: low-phosphorus will increase hyphal growth and branching as well as increase plant exudation of compounds that encourage hyphal growth.
• Chop up the roots and stems of mycorrhizal plants to use as inoculum for other plants: Grow quick growing expendable plants such as Bidens to use as inoculum material. Incorporate into nursery pots at least one month prior to outplanting.
• Get a soil test: Know what existing N and P levels are, as well as pH. These levels can indicate the probability of successful AM colonization.
• Encourage the growth of fungus by the use of mulch, dead branches, and tree logs in the landscape: Saprophytic fungus can benefi t soil, protect plants from pathogens, and indirectly help the establishment of mycorrhizal fungus.
• Use a high diversity and high density of plants: and get plants from multiple sources.
• Create dense planting “islands” : within larger restoration areas, to act as inoculatoin sources for future expansion.
8 PLANT MICROBIOME HHF Planners - Updated June 2015
Invasive plant species often are less dependent and less
supportive of mycorrhizal associations, thus reducing the
presence of mycorrhizae in surrounding soils. Research has
indicated that some invasive weeds can release compounds
into the soil that can disrupt the indigenous microbial
communities through an antimicrobial eff ect.
Also, as a fi nal insult to the native ecosystem, the eradication
of non-native trees, such as Albizia, through quick acting
poison, can result in very high nutrient loading of the
ecosystem area from the sudden drop of a large quantity of
nitrogen rich leaf litter. This sudden nutrient loading could
act as a temporary boost to the growth of other noxious
weeds. This quick nutrient loading might be worth the
sacrifi ce in the long run, to get the nutrient cycling back to
native conditions as soon as possible.
The soils at low elevation forests and urban areas in Hawai‘i
are heavily disturbed. Vegetation in these areas is almost
exclusively non-native plants and invasive weeds that have
diminished important native soil microbes.
The ability of AMF to reduce plants’ external P requirements
has an important environmental benefi t. High levels of
P in soils can result in pollution of bodies of water when
eroded soil rich in P is deposited in them. P enrichment
of water bodies causes eutrophication due to excessive
development of algae, cyanobacteria, and aquatic plants,
and this condition impairs the usefulness of these waters.
When plants rely on AMF association rather than heavy P
fertilization, risks to water quality is reduced. Arbuscular
mycorrhizal fungi, therefore, are an important component
of nutrient management programs that aim to reduce
environmental pollution. 10 Also, high soil-phosphorus
levels can lead to plant defi ciencies in other micronutrients
that have mycorrhizal-mediated uptake, such as copper.
The presence of coarse woody debris that are slow to
decompose would have been abundant in the old growth
forests of prehistoric Hawaii. Research shows that plants
such as ‘Ohi‘a lehua, Koa, and even native ferns, produce
leaf litter and wood debris that are signifi cantly slower
to decompose than most invasive plants. Leaving logs
and tree stumps of hard wood trees in reforestation areas
could be of greater benefi t than applying fi nely chopped
wood mulch, as this would better mimic the natural forest
ecosystem with a gradual breakdown of organics that will
enhance and encourage a wider colonization of benefi cial
mycorrhizal fungus. 7 The action of the mycelial networks
of saprotrophic mushrooms can help to redistribute soil
nutrients making them more available for uptake by the
mycelia of mycorrhizal fungus.
Mulch should be applied as coarse raw shredded wood
of trees, preferably of more hard wood species, such as
eucalyptus or monkey pod, rather than fi nely chopped
mulch or mulch from soft wood species such as Gun
Powder trees, Brassaia, or Albizia. Recent research indicates
that some mycorrhizae species are better at protecting
plants from pathogens, and other species are more effi cient
at helping plants obtain phosphorous 11, thus a variety of
mycorrhizae, and not just one target species, should be
considered for inoculation. Studies have shown very strong
evidence that AM fungus have great potential to protect
plants from fusarium pathogen, Pythium, verticillium wilt,
and phytophthora, as well as root-feeding nematode
infection. Mycorrhiza lowers the pH in the rhizosphere.
SOIL MICROBES IN NATIVE FORESTS VS. NON-NATIVEStudies of a site in Kohala Forest Reserve, Hawai‘i, indicate
that soil bacteria, fungi, and archaea had the highest
richness and greatest number of unique terminal restriction
fragments (micro-organisms) in ‘Ohi‘a forests, while
Eucalyptus forests and pasture had the lowest richness, and
with a unique composition. Soil pH was the best predictor
of soil microbial variation. 6 Eucalyptus had a microbial
community that was most similar to deforested pasture.
Both have high pH surface soils. Increased nitrogen in soil
and leaf liter of the nitrogen fi xing C.equisetifolia did not
Figure 3: A mono-culture forest of invasive Eucalyptus trees shifts the soil ecology away from a native endomycorrhizal soil ecology, limiting the ability for re-establishment of native plants.
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 9
result in a more distinctive microbial community.
Although the overall microbial community may be similar to
that of C.equisetifolia, C. japoinica, and A. columnaris, ‘ohi‘a
harbors and supports microbial species that are lost under
the introduce species.
Soils that are nutrient rich, such as those that have been
invaded with non-native plants that have a high rate
of nutrient cycling, can have a reduced mycorrhizal
colonization. 6
INOCULUMThe process of growing plants in nurseries in containers
does not allow for root to root inoculation of seedlings,
thus nursery grown plants are dependent on wind blown
spores unless artifi cially inoculated. Only a limited number
of mycorrhizae fungus species are capable of a reliable
dispersion of wind blown spores.
The use of mycorrhizae inoculum can help restore and
accelerate the establishment of mycorrhizae in disturbed
soils and nursery grown plants. Colonization of AM fungi
can occur from three main sources of inoculum: spores,
infected root fragments, and hyphae (mycelium fragments).
In nature, wind, water, animals, and insects can be vectors
for spreading mycorrhizae. For restoration of disturbed soil,
fungal inoculum can be prepared and applied to nursery
plants prior to outplanting, to plants at time of outplanting,
and to established plants. 18 The key to successful
inoculation is to get the spores or mycelia fragments close
to the root zone.
See Use of Mycorrhizae in Restoration of Hawaiian Habitats,
J.N. Gemma and R.E. Koske.
• Fragments of roots: Hyphae deteriorate once separated
from the host plant, but continue for weeks or months as
active propagules for use as an inoculum.
• Spores: can survive for up to 6 months to a year.
• Living Mycelium. Mycelium fragments can be added to
the soil.
• Mycorrhizal container plants or salvaged wild plants
• Topsoil from root zone of known mycorrhizal hosts
• Spores can be added to irrigation.
• An optimum level of Phosphorus in soil for the
eff ectiveness of AMF is about 0.02 mg/L or less. If P is very
low, then AMF might over compete for what it needs and
take from the plant. If P level is 0.2mg/L or higher, then
only very highly mycorrhizal-dependent species respond
signifi cantly to mycorrhizal colonization. 6 Concentrations
of P at 10mM inhibited both hyphal and branching of
mycorrhizae.
• Fungi spread between 1.5 to 3 feet per year on their own.
• Speed of colonization is more a factor of roots growing
to the inoculum rather than the inoculum growing to the
root.
• Hydroseed the inoculum, then till into soil.
• Compost tea: Mycorrhizal propagules settle out quickly
in water and must be continuously agitated to remain
in suspension, thus compost tea has limitations as an
inoculation method.
• Natural colonization depends largely on wind-blown or
water washed spores landing on each plant. Insects such
as ants are also known to transfer spores.
Inoculation by AssociationA simple method for inoculation is to place potted plants
in among an existing restored area that has already been
re-colonized by mycorrhizae and endophytic fungus. Leave
the potted plants in this “wild nursery” for a few weeks prior
to out-planting to new areas.
MAKING INOCULUM 5
1. One gallon container with drainage to produce a “pot
culture”.
2. Growing medium is quartz or basalt sand (construction
blue sand) or sterile sandy soil. Do not use peat. Sand
helps moderate a low pH and makes root removal
easier later.
3. Collect fi ne roots or soil from root zone of native
vegetation likely to be mycorrhizal. The top 10 cm of soil
will be the most colonized.
4. Add 10 to 30% collected soil or 1 cup of fi ne roots per
gallon of sand growth medium and mix thoroughly.
5. Sow host plant seeds in the container. 4-6 plants.
6. Use low phosphorus fertilizer.
7. Grow for about 3 to 4 months, then let pot dry slowly
and completely for 2-3 weeks.
8. Remove the above ground portion of the host plant
and discard.
9. Chop the roots and mix them in with sand. This root
and sand mixture is your concentrated AM fungal
inoculum. It will remain eff ective for at least one year if
dried to less than 5% moisture (dry in oven at 60c) and
stored in plastic containers and kept in a cool, dry area.
10. Create more “pot cultures” using this inoculum.
10 PLANT MICROBIOME HHF Planners - Updated June 2015
11. Use to inoculate non-mycorrhizal plants ready for out-
planting.
12. Broadcast inoculum over planting areas. Focus on base
of seedlings to maximize aff ect with less product. 5
Note that AMF development is more favorable when soil moisture is slightly less than optimal for plant growth, and soil temperature is slightly higher than optimal. It is important to select AM fungi appropriate for a target
plant. Most fungi are generalists, being willing to colonize
most any plant, but many plants are selective for only very
specifi c fungal symbionts. AM inoculum currently has
to be grown in symbiosis with living plants. This makes
them challenging to produce in large quantities through
commercial suppliers, however recent progress has been
made in commercial production. One promising new
approach encapsulates highly mycorrhizal root fragments
into beads by encapsulating them in a polymer made from
algae, enabling them to have a shelf life of at least 3 years.
For Hawaii native restoration projects, the use of non-Hawaii
sources for inoculum is questionable and should be avoided
unless the AM fungi source is certifi ed to be native to the
general area it is being used and is known to infect the
target plants.
A more practical method of inoculation of large areas of
soil with AM fungus for native plant restoration in Hawaii
could be the technique of encouraging and enhancing the
colonization of indigenous populations of fungus already
marginally present within the soil of the restoration site.
This is achieved by the extensive use of highly mycorrhizal
groundcover plants that will pre-establish a well-colonized
mycelia network that will quickly infect other native plants
within or adjacent to these plantings.
Refer to the College of Tropical Agriculture and Human
Resources (CTAHR) for an excellent pamphlet “Manual on
Arbuscular Mycorrhizal Fungus Production and Inoculation
Techniques” for additional information on mycorrhizal
inoculation in Hawai‘i. http://www.ctahr.hawaii.edu/oc/freepubs/pdf/SCM-5.pdf
LaboratoriesSoil Foodweb Incorporated of Corvallis, Oregon (541/752-
5066)
http://mail.notionsofnow.com/lab-services.html
Plant Genera in Hawai‘i that are NOT Mycorrhizal or very low Mycorrhizal:
* Most native Hawaiian plants are endomycorrhizal. The following plants are some of the exceptions to that, with low or no mycorrhizal associations:
Sesumvium/Portulaca
Cyperus
Notorichiu
Santalum
Brassiica
Rumex
Capparis (Maiapilo)
Gahnia
Fimbristylis
Carex
Phlebodium a. (Laua‘e Hoale)
Phymatosorus s. (Laua‘e)
Nephrolepis spp. (Sword Fern)
Grevellia
Plants in Hawai‘i that are Ectomycorrhizal: Papala kepau (Pisonia sandwicensis)
Māmane (Sophora chrysophylla)
Mycorrhizal Tendencies: There are no signifi cant
diff erences in AM colonization between closely related and
exotic plant species. The best predictor of colonization is
the family to which a particular species belongs. 3 Thus, if an
indigenous Hawai‘i species is mycorrhizal elsewhere in the
pacifi c or on the mainland, the same species in Hawai‘i will
likely be mycorrhizal here.
Process of Inoculation of AM fungi
1. Spore germination in soil (enhanced by appropriate host plant exudates, pH levels, and low phosphorus levels)
2. Hyphal growth fi nds a root (enhanced by root exudates and low phosphorus).
3. Host recognition (plant allows attachment and penetration).
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 11
UNDERSTANDING ENDOPHYTIC FUNGIEndophytic fungi live between plant cells in leaf and stem
plant tissue. Endophytic fungi are diff erent than mycorrhizal
fungi, but also seem to play a very important role in the
health and viability of native plants. Mycorrhizal fungus
inhabit the plant roots and form root like extensions into
the surrounding soil, which amplify the surface area of roots
to absorb nutrients, where-as endophytic fungi reside in
plant tissue throughout the plant, not just the root. Within
a few weeks of germination and leaf emergence from a
seed, a wide variety of endophytic fungi might already have
colonized the seedling. Endophytes have been found on
nearly all species of plants around the word. Aerial dispersal
in wind or by insects is the common dispersal mechanism
for the fungi.
The composition of endophytic fungi between diff erent
species of native plants is signifi cant. Native plant species
attract and harbor unique sets of endophytic fungi that are
presumably most useful to that species. Specifi c species
of Endophytic fungi of native plants may have co-evolved
with the plant, and might be essential for the health of the
plant, and an integral part of a plant biome and surrounding
native ecosystem.
The benefi ts of endophytic fungi are similar to mycorrhizal
fungi, and include:
• Increased shoot/root growth
• Enhanced pathogen and insect resistance
• Improved draught tolerance
• Enhanced nitrogen effi ciency
• Enhanced fl owering and seed germination
Understanding the role of endophytic fungi
in plants is a promising new frontier of
science that is just beginning to be unwoven.
The technology of DNA analysis is only just
now making possible the identifi cation and
understanding of endophytes a practical
area of research. The future of agriculture
and of native ecosystem restoration will
be greatly eff ected by the ongoing and
future research of the world of endophytes
and their critical role in plant biomes.
Spreading Endophytic Fungi: A simple and practical
approach to colonizing new plantings with an appropriate
mix of native endophytic fungi could be to collect leaf litter
from existing plants from intact native ecosystems and
spread the leaf litter beneath the new plantings. It’s been
shown that many endophytic fungi complete their life cycle
in dying leaves, producing spores that would be moved in
the wind and would naturally inoculate nearby trees. In the
area to be colonized, place a cluster of small branch cuttings
with intact leaves in an open jar of water as an “endophyte
bouquet”. This will help to keep the leaves hydrated while
they die to facilitate spore growth and dispersal.
Alternatively, place leaves in a zip-lock bag and store for
several weeks prior to placing in the landscape. The natural
moisture in the leaves will enable sporulation of endophytic
fungi and epiphytic fungus
When spreading endophytic fungus, care should be taken
to prevent the spread of plant pathogens. For example,
one concern would be the spread of ohi’a wilt fungus
(Ceratocystis fi mbriata) to areas current not infected. Leaf
material should not be distributed from sources know to
have pathogyen and insect infestations that are not already
present in the target area.
Figure 4: Endophytic Fungi within leaf tissue.
12 PLANT MICROBIOME HHF Planners - Updated June 2015
RHIZOBACTERIA and other SOIL
BACTERIAIt is well known that rhizobacteria are vital for nitrogen
fi xing plants, such as Koa, to fi x nitrogen. Studies have
shown that certain species of rhizobacteria also are linked
with the protection of plants from root diseases.
Rhizobacteria and mycorrhizal fungus have important
synergistic relationships. The presence of mycorrhizal
fungus enhances the activity of rhizobacteria, and the
presence of rhizobacteria increases chemical signals
involved in mycorrhizal symbiosis. AM fungus release a
chemical factor which can help activate the process of
nodulation of rhizobacteria.
Plants and microbes can have mutual benefi cial
relationships. By releasing certain amino acids, plant roots
can attract specifi c soil microbes. New research indicates
that plant roots can actively take up and digest microbes as
a source of nutrients, such as nitrogen.
Rhizobacteria nodules taken from the roots of existing well
established Koa trees can be used to make inoculum for
new Koa tree seedlings.
PLANT VIRUSES:
In regards to native ecosystems, the role of plant viruses
are little understood, but recent research indicates that
viruses might play important roles in the plant biome and
ecosystem dynamics. Plant viruses might be impacting
species invasion, interactions between plants, and the
attractiveness of plants to insects. Some viruses may
provide benefi ts to their plant hosts, such as improved heat
or cold resistance, or insect resistance, while other viruses
are clearly detrimental.
There are two basic types of viruses, acute and persistent.
Acute viruses are passed from plant to plant, usually
through insect vectors, and often cause symptomatic
problems to plant health. Persistent viruses are passed
by plants to off spring via infected seeds, rather than
from one plant to another. Persistent viruses rarely cause
recognizable adverse symptoms and might actually be the
most common virus in native plant ecosystems. 34
Much research has been done on detrimental viruses in
crop plants, such as the mosaic virus or necrotic spot virus.
Recent studies have shown that some viruses can make
plants more attractive to insects, so that although the virus
does not seem to hurt the plant directly, it will indirectly
aff ect plant health.
The propagation of native plants through multi-generations
of cuttings (cutting of a cutting of a cutting, etc) could
lead to the accumulation of detrimental viruses, as each
generation accumulates a few new acute viruses that then
persist and are passed on through cuttings, with each
generation adding a few more new acute viruses. The end
result may be a decrease in plant vigor with every new
generation removed from the original seed grown mother
plant. This might have implications for how native plants
are propagated.
Much more research is needed in the fi eld of plant viruses
to better understand the implications and roles of viruses
on native plant ecosystems.
SOIL NUTRIENTS
For any restoration project in Hawaii, an important initial
step should be to take representative soil tests. Of primary
interest is the levels of Phosphorus and pH.
WHY PHOSPHORUS AND SOIL pH MATER: Soils with high pH or high levels of phosphorus will be
problematic for restoration in terms of restoring a native
soil ecology. In general, endemic Hawaiian plants have
evolved to tolerate soils with low pH and low levels of
phosphorus by developing partnerships with mycorrhizal
fungus and bacteria. Soils with high pH or high phosphorus
can be diffi cult to re-colonize by important native soil
microbes. These microbes can be essential to the success
of a native plant in that they also represent the plants
immune system for pathogens and can be an important
source of micronutrient uptake. Without them a native plant
may have limited success. Under conditions of high soil
phosphorus, mycorrhizal fungus can sometimes actually
take phosphorus from a host plants, rather than provide it,
which ironically can lead to phosphorus defi ciency in the
host plant.
Figure 5: Seal leaves in plastic bag to create an “endophyte potpourri” to encourage sporulation. Disperse in landscape after two to three weeks .
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 13
It is important to understand the role of phosphorus availability for successful native plant restoration efforts. Keep in mind the following facts:• Hawaiian soils have very limited availability of phosphorus
for direct uptake by plant roots. This is due to iron rich
volcanic soils, warm temperatures, and low soil pH.
• Native plants have evolved to form important benefi cial
associates with mycorrhizal fungus in part to help them to
absorb phosphorus from native soils.
• Plants growing in soil with high available phosphorus
levels will eliminate colonization of mycorrhizae.
• Low phosphorus availability is a GOOD thing for the
restoration of native plants, as it requires them to form
strong mycorrhizal associations, which benefi ts them
by providing an enhanced immune system from plant
pathogens, disease, and insects, and also enhances
uptake of some critical micro-nutrients.
• Quickly decomposing organic matter, such as that from
many invasive plant species, provides excessive levels
of phosphorus for direct absorption by plant roots, thus
reducing mycorrhizal colonization.
• Standard soil tests should be conducted of site soils
to determine phosphorus levels and pH levels. By
understanding the phosphorus levels and knowing the
plant species, it is possible to predict the outcome of AM
colonization eff orts.
The more aggressive and common invasive plants in Hawaii
tend to produce leaf litter that decomposes at a much faster
rate than the leaf litter from the dominant native species.
This rapid decomposition from invasive leaf litter releases
much larger quantities of N and P into the soil, which can
further favour invasive plants, creating a feedback cycling
that can enable invasives to displace native species. 35
Why a slow decomposition of organic matter is important in
Hawaiian soils: The quick decomposition of organic matter
can increase the availability of phosphorus for plants. That’s
not good for most native plants because it reduces the
dependency on mycorrhizal fungus for phosphorus uptake,
with means reduced colonization of MF and thus reduced
benefi ts such as pathogen resistance and micro-nutrient
uptake.
Soil Test ResultsMycorrhizae colonization of plant roots is greatest when
soil phosphorus levels are at or below 50 ppm (50 mg kg-1).
Little to no colonization occurs when P is above 100 ppm.
SOIL AMENDMENTS In some cases, the application of soil amendments or
specifi c nutrients can be of benefi t to the establishment of
new plantings. After out planting, avoid using fertilizers that
are high in nitrogen or phosphorus.
During nursery production of native plants, the application
of complete fertilizers, including nitrogen and phosphorus,
is unavoidable. Nursery plants need rapid growth with
robust roots systems so that plants can be ready quickly
for effi cient and economical production. Applications of
fertilizer to nursery plants should be tapered off a few weeks
prior to outplanting.
Biochar: Made from organic material that is processed
under high heat and low oxygen. Because of its porous
structure and high surface area, Biochar might help to
enhance the activities of soil microorganisms and help to
retain nutrients for use by plants. It can also help to fi lter
heavy metals, pesticides, and other pollutants that might
otherwise migrate into the wetland and streams. Biochar is
applied by mixing into the top few inches of soil.
Zinc and Iron: High levels of phosphorus can induce a zinc
defi ciency by either precipitation of zinc or by interfering
with zinc metabolism within plant cells. In soils with high
phosphorous levels, the application of foliar zinc and iron
can be benefi cial to native plants, and will not adversely
aff ect soil microbes.
The application of chelated iron can be of benefi t to potted
or newly planted Sandalwood trees (Santalum spp.).
Humic Acid: Humifi ed organic matter (Humic Acid) is a
natural soil conditioner that acts as an organic chelator and
microbial stimulator. It enhances the plant’s ability to take
in essential nutrients and improves soil structure. Only small
quantities of humic acid are required for application.
Humic acid might enhance the microbial rhizosphere to
the benefi t of native plant restoration in disturbed soils.
Humic acid has been found to help plant growth and
suppress disease by stimulating benefi cial soil microbes
and mycorrhizae, presumably by improving absorption
of soil nutrients (iron, copper, zinc, and manganese), and
enhancing metabolic reactions. 23, 24
HAWAI‘I NATIVE PLANT MICROBIOME MANUAL
PLANT MICROBIOME 15
REFERENCES1. Bill Garnett, Wiliwili Hawaiian Plants, restoration
techniques, LICH Conference, 2013
2. The Instant Expert Guide to Mycorrhiza, the connection
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3. Do Closely Related Native and Exotic Fern Species Diff er
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Family Ties and Plant Invasions: 2012
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Kniffi n, 2008
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Fight?, Judith D. Schwartz, Yale Environment 360, Yale
School of Forestry & Environmental Studies, March 2014
16 PLANT MICROBIOME HHF Planners - Updated June 2015
26. Planting ‘Tree Island’ Helps to Facilitate Tropical
Forest Recovery, Karen D. Holl, Society for Ecological
Restoration, SER News, vol 28, issue 1, March 2014
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growth of diptoerocarp seedlings, Francis Q. Brearley,
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