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.


PLANT MICROBIOME 3


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.


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.


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.


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.


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.


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.


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).


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.


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 .


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


PLANT MICROBIOME 15

REFERENCES1. Bill Garnett, Wiliwili Hawaiian Plants, restoration

techniques, LICH Conference, 2013

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for functional Ecosystems , Ted St. John, Ph.D.: ., April

2000, http://green-diamond-biological.com/wp-content/uploads/2012/03/Mycorrhiza-Primer.pdf

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Family Ties and Plant Invasions: 2012

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Kniffi n, 2008

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hawai‘i native plant microbiome manual

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