1. Arbuscular Mycorrhiza in a Sustainable World
CSA at Plant Canada, St.Marys U, Halifax, July
C. Hamel*, N. Bazghaleh, M. Dai, E. Furrazola Gomez, Y. Torres-Arias, and A. K. Singh

2. P cycling through agricultural land2

3. P cycling through agricultural land
USA
4.6 Gt
South
Africa
4.0 Gt
Other Countries
12.6 Gt
China
14.1 Gt
Morocco and
Western Sahara
26.7 Gt P P
Gilbert, 2009 Nature P P 3

4. P cycling through agricultural land4

5. Outline
How AM improve nutrient use efficiency? Uptake Growth promotion How to enhance AM efficacy in agriculture? Genotypes Inoculation Cropping system Reduced tillage No summer fallow

6. Outline
How AM improve nutrient use efficiency? Uptake Growth promotion How to enhance AM efficacy in agriculture? Genotypes Inoculation Cropping system Reduced tillage No summer fallow

7. Arbuscular mycorrhizal fungi
AM fungi live in symbiosis (live together) with plant roots….

8. Arbuscular mycorrhizal fungi
AM fungi have an intraradical and an extraradical phase

9. Koske & Polson, 1984, BioScience
Arbuscular mycorrhizal fungi
Koske & Polson, 1984, BioScience
AM fungi are symbiotic soil organisms that improve the absorbing effectiveness of roots and, thus, plant nutrition. They also and sanitize soil rooting depth

10. Nutrient use efficiency
1. Improved nutrient recovery
2. Physiological efficiency
Experts expect little improvement in physiological efficiency of nutrient use in shoots because a lot of work has been done on this already, so futher improvement is difficult. Large improvement in efficiency of uptake is expected because not much as been done so far on this.
However, AMF influence neg 1 and 4, and positively 3 and 5, thus we do not know what to expect in selecting genotypes for better AM symbiosis
Photoinhibition (no5) occurs when photosystem II (especially) is destroyed when photon absorbed by the photosynthetic apparatus exceed their utilisation in C assimilation. It results in a reduced photosynthetic capacity.
L. Ortiz-Ribbing

11. The AM symbiosis 1. Improves nutrient recovery
2. Plant physiological efficiency
Experts expect little improvement in physiological efficiency of nutrient use in shoots because a lot of work has been done on this already, so futher improvement is difficult. Large improvement in efficiency of uptake is expected because not much as been done so far on this.
However, AMF influence neg 1 and 4, and positively 3 and 5, thus we do not know what to expect in selecting genotypes for better AM symbiosis
Photoinhibition (no5) occurs when photosystem II (especially) is destroyed when photon absorbed by the photosynthetic apparatus exceed their utilisation in C assimilation. It results in a reduced photosynthetic capacity. 11
L. Ortiz-Ribbing

12. (Yang et al., 2010, Microb Ecol)
Nutrient recovery
AM fungi are symbiotic soil organisms that improve the absorbing effectiveness of roots and, thus, plant nutrition. They also and sanitize soil rooting depth

13. Physiological efficiency
Nutrient distribution in structural and photosynthetic parts
Efficiency of N and P remobilization into grain
Plant architecture (sterile tiller, seed filling, light capture)
Delayed senescence
Tolerance to stress (water, disease, temperature, and high- light)
Experts expect little improvement in physiological efficiency of nutrient use in shoots because a lot of work has been done on this already, so futher improvement is difficult. Large improvement in efficiency of uptake is expected because not much as been done so far on this.
However, AMF influence neg 1 and 4, and positively 3 and 5, thus we do not know what to expect in selecting genotypes for better AM symbiosis
Photoinhibition (no5) occurs when photosystem II (especially) is destroyed when photon absorbed by the photosynthetic apparatus exceed their utilisation in C assimilation. It results in a reduced photosynthetic capacity.
L. Ortiz-Ribbing

14. Dennett et al., 2011 J Arid Envir
AM improves plant physiological efficiency
L. Ortiz-Ribbing
Dennett et al., 2011 J Arid Envir
Non-AM AM
Nutrient distribution in structural and photosynthetic parts
Efficiency of N and P remobilization into grain
Plant architecture:(sterile tiller, seed filling, light capture)
Delayed senescence
Tolerance to stress:(water, disease, temperature, and high- light)
Experts expect little improvement in physiological efficiency of nutrient use in shoots because a lot of work has been done on this already, so futher improvement is difficult. Large improvement in efficiency of uptake is expected because not much as been done so far on this.
However, AMF influence neg 1 and 4, and positively 3 and 5, thus we do not know what to expect in selecting genotypes for better AM symbiosis
Photoinhibition (no5) occurs when photosystem II (especially) is destroyed when photon absorbed by the photosynthetic apparatus exceed their utilisation in C assimilation. It results in a reduced photosynthetic capacity.
Reduction in root to shoot ration is often found in AM plant of temperate regions the Paper
Highlights
► Arbuscular mycorrhizas were found in wild Solanum centrale (Australian Bush Tomato). ► Inoculated unfertilised seedlings in the glasshouse exhibited moderate AM formation. ► Inoculated fertilised seedlings exhibited no AM formation. ► Inoculation lowered root:shoot ratio and raised root P content without colonisation. ► AM play a significant role in S. centrale growth in low phosphorus environments.
(n = 12; bars = LSD)

15. Ruiz-Sanchez et al., 2010, J Plant Physiol
Water
Rice shoot glutathione
(mmol g-1 DW)
AM and non-AM rice plants were maintained under well-watered conditions or were subjected to moderate and severe drought stress for 15 d. After that, half of the plants from each treatment were harvested, while the other half were allowed to recover from drought for additional 25 d. The results showed that rice can benefit from the AM symbiosis and improve their long-term development after a drought stress period. In fact, at each watering level, AM plants showed about 50% enhanced shoot fresh weight as compared to non-AM plants. The AM symbiosis enhanced the plant photosynthetic efficiency under stress over 40%, induced the accumulation of the antioxidant molecule glutathione and reduced the accumulation of hydrogen peroxide and the oxidative damage to lipids in these plants. Thus, these combined effects enhanced the plant performance after a drought stress period.
(n = 12; ± SEM) AM
Non-AM
Ruiz-Sanchez et al., 2010, J Plant Physiol

16. Water
In all living organisms, glutathione (GSH) is the major low molecular weight thiol-containing compound. Glutathione exerts its antioxidant function by reaction with superoxide radicals, peroxy radicals and singlet oxygen followed by the formation of oxidized glutathione and other disulfides (Meyer, 2007). In addition, it has several important physiological functions, including the removal of toxic oxygen derivatives in the ascorbate–glutathione cycle, the induction of several enzyme activities, and it participates in sulphur metabolism and gene expression (Foyer et al., 1995).

17. Sukhada et al., 2011, Biol Contr
Arbuscular mycorrhizal fungi
Sukhada et al., 2011, Biol Contr
Month
(n = 4; ± SEM)
Fluorescence colonies of Phytophthora in papaya (cv. Surya) roots pre-colonized with BCAs after challenging with the pathogen and studied with FITC conjugated
antibodies under field conditions. GM, G .mosseae; Tri, T. harzianum; P.F, P. fluorescens; P, Phytophthora. 2–7 indicates months after challenging.

18. (Atul-Nayyar et al., 2009 Mycorrhiza)
(n = 5; LSD α = 0.05) ab b
AM fungi are symbiotic soil organisms that improve the absorbing effectiveness of roots and, thus, plant nutrition. They also and sanitize soil rooting depth c
(Atul-Nayyar et al., 2009 Mycorrhiza)

19. (Atul-Nayyar et al., 2009 Mycorrhiza)
(n = 5; LSD α = 0.05) b
AM fungi are symbiotic soil organisms that improve the absorbing effectiveness of roots and, thus, plant nutrition. They also and sanitize soil rooting depth b
(Atul-Nayyar et al., 2009 Mycorrhiza)

20. Outline
How AM improve nutrient use efficiency? Uptake Growth promotion How to enhance AM efficacy in agriculture? Genotypes Inoculation Cropping system Reduced tillage No summer fallow

21. 3 components to AM symbiosis effectiveness
Glomus monosporum 3 components: Plant genotype, fungal genotype, the place where they live

22. Not much we can do about where they live

23. AM fungal diversity in Prairie soils
Not much we can do about AMF the fungal associatesf 23

24. Include Soil AM Potential in Soil Fertility Model
$ Benefits $
Quantity?
Quality?
Soil test
AMF
Soil P supply
P fertilizer
AMF inoculant

25. NAD1 Mitochondrial maker6 10 20
100
155
Spore number 12 30
Cloned DNA (ng)
NAD1 Mitochondrial maker
Include Soil AM Potential in Soil Fertility Model
$ Benefits $
Quantity?
Quality?
Soil test
AMF
Soil P supply
P fertilizer
AMF inoculant

26. Claroideoglomus luteum Glomus sp 04 Glomus monosporum
Rhizophagus intraradices cf.
Claroideoglomus luteum
Glomus monosporum
Claroideoglomus luteum
Claroideoglomus claroideum
Glomus monosporum
Glomus sp. 02 E
Glomus sp 05
Entrophospora infrequens
Funneliformis mosseae
Rhizophagus intraradices cf.
We can describe the AM resource available
Acaulospora kentinensis
Claroideoglomus claroideum
Claroideoglomus etunicatum cf.
Claroideoglomus luteum
Glomus sp 01
Glomus sp 03
Glomus monosporum
Funneliformis mosseae
Pacispora cf. sp. 01
Pacispora cf. sp 02
Rhizophagus intraradices cf.

27. Claroideoglomus luteum Glomus sp 04 Glomus monosporum
Rhizophagus intraradices cf.
Claroideoglomus luteum
Glomus monosporum
Claroideoglomus luteum
Claroideoglomus claroideum
Glomus monosporum
Glomus sp. 02 E
Glomus sp 05
Entrophospora infrequens
Funneliformis mosseae
Rhizophagus intraradices cf.
Develop varieties compatible with the most widespread species or the most mutualistic species
Acaulospora kentinensis
Claroideoglomus claroideum
Claroideoglomus etunicatum cf.
Claroideoglomus luteum
Glomus sp 01
Glomus sp 03
Glomus monosporum
Funneliformis mosseae
Pacispora cf. sp. 01
Pacispora cf. sp 02
Rhizophagus intraradices cf.

28. For selection to be possible, genetic variation must exist.
Methods
Treatments of five durum wheat genotypes were grown in 2 L of pasteurized loamy sand containing over 7000 live propagules of Glomus intraradices or a control of sterile propagules (autoclaved). A first experiment was conducted under poor soil fertility conditions and a 2nd experiment was conducted under conditions of medium soil fertility. The loamy soil received 0.8 L of a modified Long Ashton nutrient solution in the first experiment, and medium soil fertility condition was created, in a 2nd experiment, by applying or 1.2 L of the nutrient solution. The nutrient solution was added in increasing abundance as plants grew larger. Day/night photoperiod was 16h/8h, and temperature was 15ºC/18ºC for 7 weeks, then 18ºC/21ºC until harvest. Watering was done as needed.
Roots were sampled, cleaned, stained and the level of AM colonization was measured under a dissecting microscope (Giovannetti and Mosse, 1980). Total and grain biomasses were recorded after drying at 40°C until no further reduction of weight occurred on successive dates. Vegetative tissues were ground and analyzed for their nutrient content. Response to AM colonization was expressed as: Response = (biomassAM – biomasscontrol / biomasscontrol * 100
Objective:
to define if genetic variation in AM symbiosis formation and effectiveness exists in durum wheat

29. Genotype x AM inoculation
Summary of the probabilities of effects of durum wheat genotype, AM inoculation, and their interactions, which were observed in experiments conducted under low and medium soil fertility levels (n = 5)
Low soil fertility
Medium soil fertility
Variable
Genotype
AM inoculation
Genotype x AM inoculation
Root colonization
0.0165
n/a
0.0383
Grain biomass
0.0062
0.0543 -
<0.0001
0.0356
0.0315
Straw biomass
0.0162
0.0537
Straw P
0.0896
0.0087
Straw K
0.0389
0.0480
Straw Mn
0.0213
0.0524
0.0385
Grain P
0.0553
0.0360
Grain K
Grain Mn
0.0002
0.0005
Grain Fe
0.0346
Grain Cd
0.0070
5 genotypes and 2 inoculations (commencial)
n/a, Non inoculated plants were not colonized and not considered in this analysis of variance to comply with the normality requirement of the test.

30. Inoculation
Rhizophagus intraradices

31. Wheat
3-leaf
Wheat 3-leaf stage in inoculated plot

32. Wheat
3-leaf

33. Effect of inoculation of field-grown wheat with G
Effect of inoculation of field-grown wheat with G. intraradices on shoot P and root colonization
(n = 4; bars = SEM) * *
The field experiment was conducted at the Elizabeth
Macarthur Institute of Agricultural Research,
Menangle, 45 km south of Sydney, NSW (Australia).
The field had been under grass and legume pasture 20
years ago and bare fallow for the last 20 years. The
soil was loamy, slightly acidic (pH 5.5) with a small indigenous spore population (0.2 spores g−1 dry soil) of Glomus sp. comprised mainly of Glomus mosseae (Schenck and Perez, 1990), as estimated by wet sieving and decanting (Pacioni et al., 1992). The soil had little available phosphorus (0.016 mg g−1 of soil), as analyzed by bicarbonate-extractable method (Rayman and Higginson, 1992).
(n = 4) * *
4 weeks
After planting
10 weeks
After planting
20 weeks
After planting * * * * * * * *
P fertilizer applied (kg P ha-1)
From Mohammad et al., 2004 Agric Ecosys Environ

34. Cropping systems

35. Phylogenetic classification of the AM fungi and number of clones associated with field-grown chickpea cultivars Amit, CDC Frontier, CDC Xena and CDC Anna.
The numbers in the branches are bootstrap values from 1000 iterations in a Neighbor Joining analysis. Non-significant values (<75%) were omitted.
Ellouz, unpublished

36. Relationship between the distribution of the most abundant AM fungal species and chickpea cultivars, as revealed by PCA (N = 32) -
0.4
0.2
0.0
0.6
0.8
PC1
PC2
G.etunicatum
G.lamellosum
G.sp3
P.brasilianum
P.laccatum
P.sp1
D.sp1
D.sp2
Frontier
Amit
Xena
Anna
Plant genotype selectively influence the prolifecation of AM fungal species
Ellouz, unpublished

37. Hyphal growth of Glomus clarum exposed to 25% MeOH-soluble HPLC fraction of CDC Anna Chickpea root extract, in Petri plate assay (n = 4; ± SEM)
Hyphal Length (mm)
Control 37
HPLC fraction

38. Verbruggen et al., 2010 New Phytol
Mean arbuscular mycorrhizal fungal (AMF) richness ± SEM at various sampling dates, as determined by T-RFs
The significance (P < 0.05) of pair-wise comparisons of organic vs conventional treatments is indicated by an asterisk. July 2007: maize (n = 16; t = 0.703; P = 0.501); potato (n = 10; t = 1.986; P = 0.082); September 2007: maize (n = 16; t = 2.353; P = 0.034); July 2008 (n = 10; t = 2.722; P = 0.026); September 2008 (n = 10; t = 2.595; P = 0.032).
Natural grassland
Organic
Conventional
Mean arbuscular mycorrhizal fungal (AMF) richness ± SE in grasslands (natural) and in organic (grey bars) and conventional (black bars) potato and maize fields sampled at various sampling dates (July '07, July' 08, Sept. '07 and Sept. '08). The significance (P < 0.05) of pair-wise comparisons of organic vs conventional treatments is indicated by an asterisk. July 2007: maize (n = 16; t = 0.703; P = 0.501); potato (n = 10; t = 1.986; P = 0.082); September 2007: maize (n = 16; t = 2.353; P = 0.034); July 2008 (n = 10; t = 2.722; P = 0.026); September 2008 (n = 10; t = 2.595; P = 0.032). AMF richness is assessed as the number of forward and reverse terminal restriction fragments (T-RFs) divided by two.
Verbruggen et al., 2010 New Phytol

39. Verbruggen et al., 2010 New Phytol
Average abundance of specific AMF types as a percentage of the total in each crop ⁄ management ⁄ time combination for 2007
Shading from black to white ( , >80%; , 60–80%; , 40–60%; , 20–40%; , 0–20%; , absence) indicates the relative abundance of
AMF types across sites within a specific treatment. Each AMF type is characterized by a specific terminal restriction fragment (T-RF) pair. The
last two columns of this table show the length (bp, base pairs) of the 5￠ and 3￠ end DNA fragment sizes affiliated to each AMF type (first
column). When several T-RF pairs fell within the same higher order phylogenetic group, a further identity number was added after the group
identifier (see Supporting Information Fig. S3). At the base, the percentage of all peaks covered by the depicted AMF types is given
(percentage covered). For each AMF type, significant Spearman’s q correlations with environmental variables and crop rotation indicators are
shown for July 2007, excluding the semi-natural grassland sites. OM, organic matter. *, P < 0.05; **, P < 0.01.
>80%
60-80%
40-60%
20-40%
0-20% 0%
Verbruggen et al., 2010 New Phytol

40. Soil tillage

44. Abundance of metabolically active
Seasonal dynamics of metabolically active AM hyphae associated with field-grown maize, as influenced by tillage (n = 4; LSD, α = 0.05)
250
No-till
Reduced
Conventional
200
Abundance of metabolically active
hyphae (cm cm-3)
150
100 50
Apr.
Jun.
Aug.
Oct.
Dec.
Feb.
Apr.
Jun.
Aug.
Oct.
Time (month)
1st season
2nd season
Kabir et al., 1997 Plant Soil

45. Summer fallow

46. (n = 4 for the covered treatment, n = 2 control, ± SEM)
AM fungus propagule density for soil covered with ground cover fabric to inhibit plant growth (“covered”) vs. cropped soil (“control”), as determined by MPN
(n = 4 for the covered treatment, n = 2 control, ± SEM)
Douds et al., 2010 Plant Soil

47. Outline
How AM improve nutrient use efficiency? Uptake Growth promotion How to enhance AM efficacy in agriculture? Genotypes Inoculation Cropping system Reduced tillage No summer fallow

48. Thank you