Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue1-2.Apr2000:
Scientia Horticulturae 84 (2000) 127±140
Effect of arbuscular mycorrhizal (AM) fungal
communities on growth of `Volkamer' lemon in
continually moist or periodically dry soil
M.W. Fidelibus1, C.A. Martin*, G.C. Wright2, J.C. Stutz
Department of Plant Biology, Arizona State University, PO Box 871601,
Tempe, AZ 85287-1601, USA
Accepted 23 August 1999
Abstr act
Citrus volkameriana Tan. and Pasq. (`Volkamer' lemon) seedlings were inoculated with ®ve
different communities of arbuscular mycorrhizal (AM) fungi collected from citrus orchards in Mesa
and Yuma, AZ, USA, and undisturbed North American Sonoran Desert and Chihuahuan Desert
soils. Plants were then grown in a glasshouse for four months under continually moist or
periodically dry conditions achieved by altering watering frequency so that before watering events
container soil water tensions were approximately ÿ0.01 MPa (continually moist) or ÿ0.06 MPa
(periodically dry) one half way down the container pro®le. Plants grown in continually moist soil
had greater shoot growth than plants grown in periodically dry soil. Plant P status did not limit
growth, and there was no interaction between watering frequency and AM fungal inoculum
treatments. Plants inoculated with AM fungi from the Yuma orchard soil had signi®cantly less root
dry weight and total root length, and lower photosynthetic ¯uxes than plants treated with inoculum
from the other soils. Speci®c soil respiration and an estimated carbon cost to bene®t ratio were also
higher for plants inoculated with AM fungi from the Yuma orchard soil than for plants treated with
inoculum from the other soils. The Yuma orchard inoculum was distinctive in that > 80% of the
total number of AM fungal spores were from a single species, Glomus occultum. These data showed
that root growth suppression of plants treated with the Yuma inoculum, compared with plants
treated with inoculum from all other sites, was substantial and greater in magnitude than the effect
of periodic soil drying. Suppression of root growth might have resulted from increased AM fungal
*
Corresponding author. Fax: 1-480-965-5952.
E-mail address: [email protected] (C.A. Martin).
1
Current address: Horticultural Science Department, University of Florida, PO Box 110690,
Gainesville, FL 32611-0690, USA.
2
Current address: Yuma Mesa Agricultural Center, Rt 1 Box 40M, University of Arizona,
Somerton, AZ 85350, USA.
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 1 2 - 0
128
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
activity resulting in higher carbon costs to the plant. # 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Gas exchange; Irrigation; Mycorrhizae; Soil respiration
1. I ntr oduction
The potential of arbuscular mycorrhizal (AM) fungi to enhance citrus growth
(Menge, 1983), whole plant transpiration (Levy et al., 1983), root hydraulic
conductivity (Graham and Syvertsen, 1984) and photosynthesis (Johnson, 1984)
has been well documented. Citrus are highly mycorrhizal dependent and citrus
roots under orchard condition are normally mycorrhizal (Nemec et al., 1981). AM
fungal enhancement of citrus growth has been usually attributed to enhanced
phosphorus (P) nutrition of AM plants, but only when soil P supply is limiting
(Graham, 1986). Eissenstat et al. (1993) reported a threefold increase in the
relative growth rate of citrus seedlings inoculated with Glomus intraradices
FL208 under conditions of suboptimal soil P. In contrast, Peng et al. (1993)
reported a 12% reduction in citrus relative growth rates by G. intraradices under
conditions of high soil P. Because high energy phospho-nucleotides mediate
enzymatically driven plant processes in an non-steady state manner (Eissenstat
et al., 1993), it is dif®cult to identify potential AM fungal effects on plant
function that are not directly related to mycorrhizal enhancement of plant
P nutrition. There is, however, increasing evidence that AM fungi stimulate citrus
root growth independent of P nutrition (Eissenstat et al., 1993, Graham and
Syvertsen, 1984; Peng et al., 1993). Since citrus seedlings with small root systems
are vulnerable to desiccation (Davies and Albrigo, 1994), inoculation of citrus
roots with AM fungal populations that enhance growth might improve transplant
survival of seedling nursery stock into ®eld sites.
Previous research documenting the effect of AM fungi on citrus growth and
physiology have largely been based on differences between plants inoculated with
a single common isolate of AM fungi (typically G. intraradices) and non-AM
plants. However, citrus orchard soils contain communities of AM fungi rather
than a single species (Nemec et al., 1981), and several or all of these species
might colonize citrus roots at the same time. The relevance of species diversity to
the function of AM fungi in the ®eld is poorly understood (Graham et al., 1996)
because data comparing different communities of AM fungi on plant growth and
physiology are lacking (Graham, 1986).
Different species, and even geographic isolates of the same species of AM
fungi might vary with respect to their ability to colonize roots and improve plant
growth (Camprubi and Calvet, 1996; Graham et al., 1982, 1996). Relatively high
water and nutrient soil inputs might, over time, favor proliferation of species or
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
129
strains of AM fungi colonizing citrus roots that, while tolerant of cultural
management practices, are less likely to enhance plant growth (Kurle and P¯eger,
1994). Increased below-ground carbon allocation to AM fungi colonizing plant
roots might result in plant growth depression if not compensated for by an
increase in carbon assimilation (Graham et al., 1996; Graham and Eissenstat,
1998). In this study, we compared effects of AM fungal communities from
orchard soils and undisturbed desert sites on growth and physiology of
`Volkamer' lemon grown under continually moist or periodically dry soil
conditions achieved by altering watering frequency.
2. M ater ials and methods
Communities of AM fungi from two Arizona, USA, citrus orchard soils and
from three undisturbed desert soils in the Southwestern United States were used
as inoculum in this study. AM fungal populations from citrus orchards originated
from rhizosphere soil of either `Volkamer' lemon rootstock grown at the Yuma
Mesa Agricultural Experiment Station in Yuma, AZ, USA (32.68N, 114.68W), or
from rhizosphere soil of sour orange (Citrus aurantium L.) rootstock grown at a
commercial orchard in Mesa, AZ, USA (33.28N, 111.58W). AM fungal
populations from desert locations originated from rhizosphere soil of Prosopis
spp. growing in the central Sonoran Desert near Verde River, AZ, USA (33.38N,
111.48W), the western Sonoran Desert near Borrego Springs, CA, USA (33.18N,
116.28W), or the western Chihuahuan Desert near Padre Canyon, TX, USA
(31.48N, 105.68W).
Soil from each site was used to establish trap cultures of each AM fungal
community (Stutz and Morton, 1996). The AM fungal communities from citrus
orchard soils were cultured for one cycle (3.5 months) on sudan grass (Sorghum
sudanese [Piper.] Staph.). Desert populations were cultured for three cycles on
sudan grass to increase the AM fungal inoculum potential (Stutz and Morton,
1996). Colonized root pieces, hyphae, rooting substrate, and spores from the trap
cultures were used as inoculum. The AM fungal species composition of each
inoculum was determined by extracting spores from a 100 cm3 subsample via wet
sieving, decanting and sucrose gradient centrifugation (Daniels and Skipper,
1982). A dissecting microscope was used to count spore morphotypes. Each
morphotype was mounted in PVLG and Meltzer's/PVLG (Koske and Tessier,
1983) and identi®ed based on subcellular characteristics.
Fifty uniform (0.10 m tall), two-month old, non-mycorrhizal `Volkamer' lemon
seedlings were potted into 8 l plastic pots ®lled with a pasteurized potting mixture
of coarse sand (particle size < 4.25 mm) blended with soil (3 : 1 by volume)
which had been passed through a 2 mm sieve. The soil was a Gilman clay loam
collected at a depth of 0.1±0.3 m in the A horizon from the Arizona State
130
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
University Horticultural Resource Unit, Tempe, AZ, USA. Soil analysis was as
follows: pH 7.3, EC 0.25 dS/m, 11.26 mg sodium bicarbonate extractable P/g
soil, and 13.2 g organic matter per kilogram soil. During the study all plants were
grown in a temperature-controlled glasshouse under natural irradiance (air
temp 308C day/218C night, mean maximum PAR 1200 mmol/m2/s, VPD
range 1 to 2 KPa).
At potting, inoculum from one of the ®ve AM fungal populations was placed
subjacent to the transplants. Inoculum potential was previously determined using
the most probable number method (Alexander, 1982), and each pot received
approximately 1.5 106 AM fungal propagules. All plants were fertilized with
12 g of 20N±0P±16.6 K±2Fe±1.4 Mn (slow release, N source as isobutylidene
diurea) and 22 g of 0N±19.6P±0K±12S (concentrated superphosphate).
After a one-week acclimatization period, half of the plants were randomly
assigned to either a continually moist or periodically dry watering treatment.
Over a four-month period, plants subjected to the continually moist treatment
were watered in excess of container capacity when soil matric potential (water
tension) decreased to ÿ0.01 MPa (approximately a 3-day cycle). Plants subjected
to the periodically dry treatment were watered in excess of container capacity
when potting soil water tensions fell to ÿ0.06 MPa (approximately a 12-day
cycle). Soil water tension was monitored with tensiometers (Soilmoisture
Equipments, Santa Barbara, CA, USA) inserted into the soil so that the ceramic
tips were located approximately one-half the distance between the surface and the
container bottom and 4 cm from the container wall.
Approximately three months after the initiation of watering frequency
treatments, leaf gas exchange and water potential measurements and soil
respiration measurements were made on the second day after an irrigation event
when soil water tension and plant water status were nearly uniform in all
treatments. Leaf gas exchange measurements were made at mid-day on recently
expanded leaves of similar appearance (approximately the seventh leaf from the
stem apex) on each plant using an infra red gas analyzer (IRGA, LI-COR 6200;
LI-COR, Lincoln, NE, USA) and 0.25 l chamber attachment. A pressure bomb
(Soil Moisture Equipments, Santa Barbara, CA, USA) was used to measure predawn and mid-day leaf water potential. Each water potential measurement was
based on a single leaf per plant using recently fully expanded leaves of similar
appearance (approximately the eighth leaf from the apex). For soil respiration
measurements, polyvinyl chloride cylindrical collars (7.0 cm height, 10.1 cm
inside diameter) were inserted into the potting soil to a depth of about 2 cm one
day before the measurements were made. A soil respiration chamber (LI-COR
6000-09) attached to the IRGA was ®tted to the collar such that the chamber air
supply manifold was 1±2 cm above the potting soil surface. A foam gasket was
placed between the collar and the chamber to prevent air leaks. Soil respiration
per pot was derived from the average of four consecutive CO2 ¯ux measurements.
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
131
At the end of the study, total stem length was measured and all plants were
harvested. Leaves and stems were separated from roots. Roots were washed free
of soil and fresh root weights were recorded. Canopy leaf area and root lengths
were measured using a digital camera interfaced with a computer (AgVision
Digital Imaging System, Pullman, WA, USA). All roots were inspected optically
(with the aid of a stereomicroscope) for pathogens and symptoms of disease and
appeared to be healthy. A sample of 1 cm length root pieces (approximately 1 g
fresh weight) was collected from each tree and ®xed in a formalin±acetic acid±
alcohol solution. Roots were stained using 0.05% trypan blue in acidic glycerol
(Koske and Gemma, 1989). The magni®ed intersections method (McGonigle
et al., 1990) was used to quantify AM fungal colonization of roots. Roots and
shoots were oven dried at 658C for 48 h and dry weights were measured. An
estimation of the carbon cost to bene®t ratio (RT/AT) of plants in response to
treatments was calculated as
RT =AT RSP RDW SA= A LA;
where RSP is the speci®c soil respiration (mmol/m2/s/g root dry weight), RDW the
total root dry weight (g/plant), SA the container soil surface area (0.042 m2),
A the carbon assimilation ¯ux (mmol/m2/s), and LA is the canopy leaf area
(m2/plant).
Phosphorus concentrations in dry, pulverized leaves were determined by the
ascorbic method (Watanabe and Olson, 1965). Dry, pulverized tissue was boiled
in ethanol to remove sugars. Starch was extracted from the remaining tissue pellet
by digestion in a solution of a-amylase and amyloglucosidase. Carbohydrate
concentrations in the resulting solutions were determined using a modi®ed
anthrone method with phenol substituted for anthrone (Scott and Melvin, 1953).
The experiment was arranged in a split plot (continually moist and periodically
dry) completely randomized block design with ®ve AM fungal inoculum
treatments and ®ve replications for a total of 50 plants. All data was subjected to
the general linear model (GLM) procedure (SAS Institute, Cary, NC, USA) for
analysis of variance. Percent AM root colonization data were arcsin transformed
to approximate a normal distribution for analysis of treatment effects. Actual
percentage means were reported. Comparisons among treatments were made with
Duncan's Multiple Range Test.
3. Results
AM species composition of inoculum and mycorrhizal colonization responses.
A range of 4±7 AM fungal species were detected in the inoculum mixtures
(Table 1). The lowest number of species was detected in the Yuma orchard
inoculum while the highest number was in the Borrego Springs desert inoculum
132
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 1
Arbuscular mycorrhizal fungal species composition of Borrego Springs (BS), Padre Canyon (PC),
Verde River (VR), Mesa (ME) and Yuma (YU) inoculum
AM fungal species and authority
Acaulospora trappei Ames & Linderman
Glomus eburneum Kennedy, Stutz & Morton
Glomus etunicatum Becker & Gerdemann
Glomus intraradices Schenck & Smith
Glomus macrocarpum Tulasne & Tulasne
Glomus microaggregatum Koske, Gemma & Olexia
Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe
Glomus occultum Walker
Glomus spurcum (Pfeiffer, Walker & Bloss)
Kennedy, Stutz & Morton
Glomus versiforme (Karsten) Berch
Glomus spp. AZ112b
Relative inoculum abundance (%)a
BS
PC
VR
ME
YU
21
0
2
11
0
30
1
0
33
47
0
0
22
8
20
3
0
0
0
5
0
16
2
0
0
0
0
0
37
1
22
15
15
10
0
11
4
0
0
13
0
81
0
2
0
0
0
55
11
0
0
0
2
a
Number of AM fungal spores for each species as a percentage of the total number of spores in
a 100 cm3 sample.
b
Undescribed species number refers to accession number at International Collection of
Arbuscular and Vesicular±Arbuscular Mycorrhizal Fungi, West Virginia University, Morgantown,
WV, USA.
(Table 1). Glomus microaggregatum was the only species detected in all of the
inoculum treatments. The Yuma orchard inoculum was distinctive in that > 80%
of the total number of AM fungal spores were from a single species, Glomus
occultum.
There was no interaction effect of inoculum and watering frequency on AM
fungal colonization of plant roots. Plants exposed to periodic soil drying had less
total colonization, arbuscules, and vesicles than well-watered plants (Table 2).
There were no differences in total colonization between inoculum treatments;
however, arbuscular colonization was greatest in plants treated with AM fungi
from the Yuma orchard. Plants treated with the Mesa orchard or Verde River
desert inoculum had a similar number of vesicles and more than plants grown
with other inocula. No septate hyphae were observed in the roots, nor was any
necrosis of cortical cells observed.
Plant growth responses and tissue P levels. Plant growth was not affected by
the interaction of watering frequency and inoculum treatments, thus, we present
plant growth responses to watering and mycorrhizal treatments separately. At
harvest, plants grown in periodically dry soil had signi®cantly less total stem
length and shoot dry weight, lower shoot to root dry weight ratio (S/R), less
canopy leaf area, and a lower canopy leaf area to root length ratio (LA/RL) than
plants grown in continually moist soil (Table 3). Root dry weight, total root
133
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 2
Effect of watering frequency and inoculum treatments on percent root length colonized by AM
fungi
Treatment
Arbuscules
Vesicles
Hyphae only
Total
Watering
Continually moist
Periodically dry
31aab
23 b
15 a
3b
19 a
16 a
64 a
43 b
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
29
21
21
25
39
6b
4b
12 ab
17 a
5b
16
17
23
20
13
49
44
58
59
57
abc
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
Values are treatment means, n 25 for watering frequency treatments and n 5 for
mycorrhizal inoculum treatments.
b
Means followed by a different letter are signi®cantly different (a 0.05) according to LSD, or
c
Duncan's Multiple Range Test.
length, and leaf P concentrations were not signi®cantly affected by watering
frequency treatments.
AM fungal population treatments had similar effects on plant growth with a
few exceptions (Table 3). Plants treated with the Padre Canyon or Verde River
inoculum had greater shoot dry weight than plants treated with the Yuma orchard
inoculum. Plants treated with the Yuma orchard inoculum had lower total root
Table 3
Effect of irrigation frequency and inoculum treatments on total stem length, shoot and root dry
weights, shoot to root dry weight ratio (S/R), canopy leaf area, total root length, canopy leaf area to
total root length ratio (LA/RL) and leaf phosphorus concentration (P) of Citrus volkameriana
Treatment
Shoot dry Root dry S/R
Stem
weight
weight
length
(g/plant) (g/plant) (g/plant)
Canopy
leaf
area
LA/RL
Root
length
(m/plant)
P
Watering
Continually moist 0.70aab
Periodically dry 0.60 b
5.9 a
4.1 b
6.1 a
5.4 a
0.96 a
0.77 b
1.02 a
0.73 b
2.88 a
2.74 a
0.39 a
0.28 b
2.1 a
2.1 a
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
5.6 a
5.3 ab
5 .4 ab
5.7 a
3.3 b
6.0
6.3
6.4
6.4
3.6
0.93
0.84
0.84
0.84
0.91
0.86
0.87
0.91
1.12
0.63
2.8
3.3
3.1
3.0
1.7
0.30
0.26
0.29
0.37
0.37
2.1
1.8
1.8
2.3
2.7
a
0.59
0.66
0.71
0.77
0.51
ab
a
a
a
b
a
a
a
a
b
a
a
a
a
a
ab
ab
ab
a
b
a
a
a
a
b
ab
b
ab
a
a
b
b
b
ab
a
Values are treatment means, n 5.
Within treatments, column means followed by a different letter are signi®cantly different
according to Duncan's Multiple Range Test (a 0.05).
b
134
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
length and root dry weight than plants treated with the other inoculum treatments.
Plants treated with the Yuma orchard inoculum also had higher leaf P
concentrations than plants grown with the other inoculum treatments. Plants
treated with the Mesa or Yuma inoculum differed in canopy leaf area, with the
former having signi®cantly greater area than the latter. However, plants treated
with the orchard inocula had higher LA/RL than plants treated with the desert
inoculum from Padre Canyon. Mycorrhizal inoculum treatments had no effect on
S/R.
Physiological responses and carbohydrate analysis. There was a signi®cant
interaction between watering frequency and AM fungal inoculum treatments on
leaf gas exchange (Table 4). When grown in moist soil, plants treated with the
Padre Canyon desert inoculum had the highest leaf carbon assimilation (A) and
stomatal conductance (gs). In contrast, plants treated with the Yuma orchard
inoculum had the lowest A and gs, while plants treated with the Yuma orchard and
Verde River desert inoculum had the lowest water use ef®ciency (WUE)
compared with plants treated with other inocula. Mycorrhizal inoculum
treatments had no effect on CI : CA.
When grown in periodically dry soil, plants treated with Yuma orchard
inoculum had the lowest A and WUE, and the highest CI : CA (Table 4). Plants
treated with Borrego Springs or Padre Canyon desert inocula had the highest gs,
while plants inoculated with the Verde River or Mesa inocula had the lowest
CI : CA compared with plants treated with the other mycorrhizal inoculum
treatments.
Speci®c soil respiration (RSP) was affected by an interaction of watering
frequency and inoculum treatments (Table 4). Plants treated with the Yuma or
Mesa orchard inocula had greater RSP than other plants treated with the desert
inocula (P 0.02) when grown in continually moist soil. However, only plants
treated with the Yuma orchard inoculum had greater RSP that was between two
and three times more than plants treated with the any of the other inoculum
treatments when grown in periodically dry soil. Our estimated carbon cost to
bene®t ratio of plants treated with the Yuma orchard inoculum was about 1.7
(continually moist) or 2.4 times (periodically dry) higher than for plants treated
with the other inocula.
Leaf sugar levels were not affected by watering frequency, but leaf starch levels
were lower for plants in periodically dry soil compared with plants grown in
continually moist soil (Table 5). Plants grown in continually moist soil had higher
root sugar and starch concentrations than plants grown in periodically dry soil.
Leaf carbohydrate concentrations were not affected by AM fungal population
treatment (Table 5). Plants treated with the Borrego Springs desert inoculum had
higher levels of root sugar than plants treated with Verde River desert inoculum,
and higher levels of starch than plants treated with Verde River desert or Mesa
orchard inocula (Table 5). Watering frequency and AM fungal inoculum
135
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 4
Effect of watering frequency and inoculum treatments on leaf carbon assimilation (A), stomatal
conductance (gs), ratio of internal to ambient CO2 concentration (CI : CA), instantaneous water use
ef®ciency (WUE A/instantaneous leaf transpiration), speci®c soil respiration (RSP soil
respiration per gram of root dry weight), and the estimated carbon cost to bene®t ratio (RT/AT)
of Citrus volkameriana
A
(mmol/m2/s)
gs
(mmol/m2/s)
CI : CA
WUE
RSP
(mmol/m2/sg)
RT/AT
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
9.8adcb
11.6 a
10.3 bc
11.0 ab
8.7 d
108.4 bc
141.5 a
119.0 b
124.8 ab
97.5 c
0.54
0.58
0.57
0.54
0.55
a
a
a
a
a
4.1
4.2
3.8
4.2
3.6
a
a
b
a
b
0.16
0.12
0.12
0.23
0.23
b
b
b
a
a
0.045b
0.030b
0.032b
0.042b
0.064a
Periodically dry
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
10.5 a
10.0 a
10.4 a
10.5 a
8.7 b
114.9
113.0
100.8
100.5
104.2
0.54
0.56
0.50
0.49
0.59
b
ab
c
c
a
4.2
4.0
4.5
4.5
3.4
a
b
a
a
c
0.14
0.13
0.11
0.16
0.35
b
b
b
b
a
0.041b
0.043b
0.033b
0.046b
0.100a
NSc
***
*
***
***
***
Watering
Continually moist
Significance
Watering
Inoculum
Watering x
Inoculum
a
a
b
b
ab
NS
**
*
NS
***
**
NS
***
NS
NS
***
NS
a
Values are treatment means, n 5.
Within watering frequency treatments, column means followed by a different letter are
signi®cantly different according to Duncan's Multiple Range Test (a 0.05).
c
Non-signi®cant, or signi®cant at P 0.05(*), 0.01 (**), or 0.001, respectively.
b
treatments did not affect pre-dawn or mid-day leaf water potentials when
measurements were taken under similar (>ÿ0.01 MPa) soil water tensions (data
not shown).
4. Discussion
These data show that communities of AM fungi can mitigate substantially
different effects on growth `Volkamer' lemon plants. The mycorrhizal effects we
measured on plant growth may be the result of changes in photosynthate
production and/or partitioning (Syvertsen and Graham, 1990). Plants treated with
136
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 5
Effect of watering frequency and inoculum treatments on the concentration (% tissue dry weight) of
sugar and starch in Citrus volkameriana leaves and roots
Treatment
Leaf
Root
Sugar
Starch
Sugar
Starch
Watering
Continually moist
Periodically dry
4.88aab
4.37 a
3.10 a
2.16 b
2.33 a
1.65 b
1.41 a
0.97 b
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
4.56
4.55
5.20
4.55
4.23
2.67
2.36
2.56
2.72
2.87
2.26
2.01
1.76
1.95
1.98
1.82
1.29
0.89
0.81
1.13
ac
a
a
a
a
a
a
a
a
a
a
ab
b
ab
ab
a
ab
b
b
ab
a
Values are treatment means, n 25 for watering frequency treatments and n 5 for
mycorrhizal inoculum treatments.
b
Means followed by a different letter are signi®cantly different according to LSD, or
c
Duncan's Multiple Range Test (a 0.05).
the Yuma orchard inoculum had root systems that were 43±49% smaller than
other plants, and when averaged across both irrigation treatments also had A
¯uxes that were 18% lower, and RSP and estimated carbon cost to bene®t ratio
values that were at least 2.0 times higher than plants treated with the other
inocula. These data support previous ®ndings that some AM fungi can reduce
growth of citrus if the high carbon costs of AM roots are not offset by increased
carbon assimilation (Graham et al., 1996).
Arid soils have high AM fungal diversity (Stutz and Morton, 1996), but
intensive ®eld cultivation might select for species or isolates of AM fungi that
tolerate horticultural practices, but which are not particularly bene®cial to crop
plants in terms of increased plant growth (Kurle and P¯eger, 1994). Unlike the
other AM fungal populations we evaluated, we found that the AM fungal
population from the Yuma orchard site was dominated (based on the proportion of
spores) by a single AM fungal species, G. occultum. Johnson et al. (1992) found
that G. occultum proliferated in corn ®elds, and that spore abundance of G.
occultum correlated negatively with corn plant dry mass and yield. Graham et al.
(1996) also found that aggressive strains of AM fungi enhanced plant P uptake
and growth at low soil P, but caused growth depression of citrus at high soil P
(Graham et al., 1996). Furthermore, Modjo and Hendrix (1986) linked growth
depression of tobacco plants to mycorrhizal colonization under conditions of high
soil P.
The Yuma orchard soil was very low in organic matter and available P which
might have favored proliferation of aggressive strains of AM fungi that enhance P
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
137
uptake. Though possibly bene®cial in situ at the Yuma site, G. occultum, might
have been responsible for the poor growth performance of the P suf®cient lemon
plants treated with the Yuma orchard inoculum in our study. In support of this
hypothesis, we note that plants inoculated with AM fungi from the Yuma orchard
soil had higher leaf P concentrations and more arbuscules (sites of carbon and P
exchange) per root length than plants treated AM fungi from the other soils. This
suggests that increased AM fungal activity might have resulted in higher carbon
costs to the plant.
Arbuscular mycorrhizal fungi can induce high rates of root/soil respiration
which represent considerable carbon costs to the plant (Peng et al., 1993;
Graham et al., 1996). If the carbon sink of AM roots is not compensated for by
increased A, then growth depression will result (Graham et al., 1996). High
values of RSP might indicate that the carbon cost to bene®t ratio of the AM
association to the plant is great and/or that fungal activity is high (Eissenstat
et al., 1993; Graham et al., 1996). Thus, we conclude that the Yuma orchard
inoculum presented lemon plants with the highest carbon cost to bene®t ratio,
which might explain the reduced root length and lower root dry weights found
in those plants.
Plants treated with Yuma orchard inoculum and grown under periodically dry
soil conditions had lower A and gs than other plants, but had CI : CA that was
similar or higher than other plants. Since CI did not decrease with gs, A limitation
for these plants was at least partly stomatal (Mott, 1988). Leaf P concentrations
can also limit A (Syvertsen and Lloyd, 1994), but plants treated with Yuma
inoculum had the highest leaf P which is evidence that reduced A was not caused
by P limitation. Though leaf P concentrations for all plants were suf®cient for
normal plant growth, we did not measure levels of other nutrients which might
have limited A. Morphological differences such as increased speci®c leaf weight
or reduced root hydraulic conductance could reduce A in citrus. However, speci®c
leaf weight (data not shown) was not affected by inoculum and though root
conductance was not measured, speci®c root weight (data not shown), and
root : shoot ratio were similar among inoculum treatments, suggesting that root
hydraulic conductivity might be similar among treatments.
Although plants treated with the Yuma orchard inoculum had the highest
estimated carbon cost to bene®t ratio, they did not have signi®cantly different
levels of non-structural carbohydrates from plants treated with other inocula. In
plants with adequate P nutrition, AM fungi affect root carbohydrates more than
leaf carbohydrates (Nemec and Guy, 1982), as observed in this study. Reducing
sugars are apparently the most responsive to AM infection, but are the smallest
carbohydrate fraction, so differences in sugar and starch levels caused by AM
fungi can be subtle (Nemec and Guy, 1982; Graham et al., 1997). Since only total
sugars were measured in this study, potential small differences in reducing sugars
due to AM treatment were not detectable.
138
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Shoot growth was positively correlated to watering frequency. Plants grown
under the periodically dry soil had lower leaf carbohydrate concentrations than
plants grown under continually moist conditions. Large citrus seedlings have
greater carbohydrate concentrations than small seedlings (Nemec and Guy, 1982),
so an analysis of variance with shoot size as a covariate was done and showed that
shoot carbohydrates were not different if size was taken into account. Thus, shoot
growth was probably not limited by leaf carbohydrate concentrations in the dry
treatment, but by some other mechanism.
Shoot growth at low soil water potential can be reduced by changes in the
elastic properties of cell walls (Nonami and Boyer, 1990) or by production of
abscisic acid in the roots (Saab et al., 1990). None of the inocula showed
signi®cant effects in terms of enhancement of leaf water potential or
improvement of shoot growth. However, AM fungal inoculum did affect aspects
of plant morphology that might enhance plant water status, such as lowered leaf
area : root length or root : shoot ratios.
Root growth was not affected by soil drying, but root growth can be less
sensitive than shoot growth to soil drying (Hsiao and Jing, 1987). Lower root
carbohydrate concentrations in plants grown under periodically dry conditions
might have been a result of lower carbon acquisition in these plants since canopy
leaf area and LA/RL was signi®cantly reduced in plants exposed to periodic soil
drying.
5. Conclusion
Growth suppression of plants treated with AM fungi from the Yuma orchard,
compared with plants treated with inocula from desert soils or the Mesa orchard,
was substantial and greater in magnitude than the main effects of the watering
frequency treatments. Previous studies have reported dramatic differences in
citrus growth as a function of different mycorrhizal isolates. The causes of
differential growth can be phosphorus limitation (Graham et al., 1982; Camprubi
and Calvet, 1996) or high carbon costs (Graham et al., 1997; Johnson et al., 1997;
Graham and Eissenstat, 1998). Since P limitation was not a factor in our study,
the differences in carbon costs of the populations used probably caused the
growth effects observed. Further research is needed to determine which AM
fungal isolate or combinations of isolates are responsible for plant growth
depression. Single species from the Yuma inoculum could be isolated, and
inoculation of citrus with isolates individually and in combination would con®rm
which fungi were responsible for plant growth suppression. Analysis of the
effects of the Yuma population compared with the other inocula on growth of
citrus under P limited conditions might provide insight into management
strategies to enhance AM fungal bene®ts in citrus orchards.
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
139
Acknowledgements
The authors wish to thank the Arizona Citrus Research Council for partial
support of this work.
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Effect of arbuscular mycorrhizal (AM) fungal
communities on growth of `Volkamer' lemon in
continually moist or periodically dry soil
M.W. Fidelibus1, C.A. Martin*, G.C. Wright2, J.C. Stutz
Department of Plant Biology, Arizona State University, PO Box 871601,
Tempe, AZ 85287-1601, USA
Accepted 23 August 1999
Abstr act
Citrus volkameriana Tan. and Pasq. (`Volkamer' lemon) seedlings were inoculated with ®ve
different communities of arbuscular mycorrhizal (AM) fungi collected from citrus orchards in Mesa
and Yuma, AZ, USA, and undisturbed North American Sonoran Desert and Chihuahuan Desert
soils. Plants were then grown in a glasshouse for four months under continually moist or
periodically dry conditions achieved by altering watering frequency so that before watering events
container soil water tensions were approximately ÿ0.01 MPa (continually moist) or ÿ0.06 MPa
(periodically dry) one half way down the container pro®le. Plants grown in continually moist soil
had greater shoot growth than plants grown in periodically dry soil. Plant P status did not limit
growth, and there was no interaction between watering frequency and AM fungal inoculum
treatments. Plants inoculated with AM fungi from the Yuma orchard soil had signi®cantly less root
dry weight and total root length, and lower photosynthetic ¯uxes than plants treated with inoculum
from the other soils. Speci®c soil respiration and an estimated carbon cost to bene®t ratio were also
higher for plants inoculated with AM fungi from the Yuma orchard soil than for plants treated with
inoculum from the other soils. The Yuma orchard inoculum was distinctive in that > 80% of the
total number of AM fungal spores were from a single species, Glomus occultum. These data showed
that root growth suppression of plants treated with the Yuma inoculum, compared with plants
treated with inoculum from all other sites, was substantial and greater in magnitude than the effect
of periodic soil drying. Suppression of root growth might have resulted from increased AM fungal
*
Corresponding author. Fax: 1-480-965-5952.
E-mail address: [email protected] (C.A. Martin).
1
Current address: Horticultural Science Department, University of Florida, PO Box 110690,
Gainesville, FL 32611-0690, USA.
2
Current address: Yuma Mesa Agricultural Center, Rt 1 Box 40M, University of Arizona,
Somerton, AZ 85350, USA.
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 1 2 - 0
128
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
activity resulting in higher carbon costs to the plant. # 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Gas exchange; Irrigation; Mycorrhizae; Soil respiration
1. I ntr oduction
The potential of arbuscular mycorrhizal (AM) fungi to enhance citrus growth
(Menge, 1983), whole plant transpiration (Levy et al., 1983), root hydraulic
conductivity (Graham and Syvertsen, 1984) and photosynthesis (Johnson, 1984)
has been well documented. Citrus are highly mycorrhizal dependent and citrus
roots under orchard condition are normally mycorrhizal (Nemec et al., 1981). AM
fungal enhancement of citrus growth has been usually attributed to enhanced
phosphorus (P) nutrition of AM plants, but only when soil P supply is limiting
(Graham, 1986). Eissenstat et al. (1993) reported a threefold increase in the
relative growth rate of citrus seedlings inoculated with Glomus intraradices
FL208 under conditions of suboptimal soil P. In contrast, Peng et al. (1993)
reported a 12% reduction in citrus relative growth rates by G. intraradices under
conditions of high soil P. Because high energy phospho-nucleotides mediate
enzymatically driven plant processes in an non-steady state manner (Eissenstat
et al., 1993), it is dif®cult to identify potential AM fungal effects on plant
function that are not directly related to mycorrhizal enhancement of plant
P nutrition. There is, however, increasing evidence that AM fungi stimulate citrus
root growth independent of P nutrition (Eissenstat et al., 1993, Graham and
Syvertsen, 1984; Peng et al., 1993). Since citrus seedlings with small root systems
are vulnerable to desiccation (Davies and Albrigo, 1994), inoculation of citrus
roots with AM fungal populations that enhance growth might improve transplant
survival of seedling nursery stock into ®eld sites.
Previous research documenting the effect of AM fungi on citrus growth and
physiology have largely been based on differences between plants inoculated with
a single common isolate of AM fungi (typically G. intraradices) and non-AM
plants. However, citrus orchard soils contain communities of AM fungi rather
than a single species (Nemec et al., 1981), and several or all of these species
might colonize citrus roots at the same time. The relevance of species diversity to
the function of AM fungi in the ®eld is poorly understood (Graham et al., 1996)
because data comparing different communities of AM fungi on plant growth and
physiology are lacking (Graham, 1986).
Different species, and even geographic isolates of the same species of AM
fungi might vary with respect to their ability to colonize roots and improve plant
growth (Camprubi and Calvet, 1996; Graham et al., 1982, 1996). Relatively high
water and nutrient soil inputs might, over time, favor proliferation of species or
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
129
strains of AM fungi colonizing citrus roots that, while tolerant of cultural
management practices, are less likely to enhance plant growth (Kurle and P¯eger,
1994). Increased below-ground carbon allocation to AM fungi colonizing plant
roots might result in plant growth depression if not compensated for by an
increase in carbon assimilation (Graham et al., 1996; Graham and Eissenstat,
1998). In this study, we compared effects of AM fungal communities from
orchard soils and undisturbed desert sites on growth and physiology of
`Volkamer' lemon grown under continually moist or periodically dry soil
conditions achieved by altering watering frequency.
2. M ater ials and methods
Communities of AM fungi from two Arizona, USA, citrus orchard soils and
from three undisturbed desert soils in the Southwestern United States were used
as inoculum in this study. AM fungal populations from citrus orchards originated
from rhizosphere soil of either `Volkamer' lemon rootstock grown at the Yuma
Mesa Agricultural Experiment Station in Yuma, AZ, USA (32.68N, 114.68W), or
from rhizosphere soil of sour orange (Citrus aurantium L.) rootstock grown at a
commercial orchard in Mesa, AZ, USA (33.28N, 111.58W). AM fungal
populations from desert locations originated from rhizosphere soil of Prosopis
spp. growing in the central Sonoran Desert near Verde River, AZ, USA (33.38N,
111.48W), the western Sonoran Desert near Borrego Springs, CA, USA (33.18N,
116.28W), or the western Chihuahuan Desert near Padre Canyon, TX, USA
(31.48N, 105.68W).
Soil from each site was used to establish trap cultures of each AM fungal
community (Stutz and Morton, 1996). The AM fungal communities from citrus
orchard soils were cultured for one cycle (3.5 months) on sudan grass (Sorghum
sudanese [Piper.] Staph.). Desert populations were cultured for three cycles on
sudan grass to increase the AM fungal inoculum potential (Stutz and Morton,
1996). Colonized root pieces, hyphae, rooting substrate, and spores from the trap
cultures were used as inoculum. The AM fungal species composition of each
inoculum was determined by extracting spores from a 100 cm3 subsample via wet
sieving, decanting and sucrose gradient centrifugation (Daniels and Skipper,
1982). A dissecting microscope was used to count spore morphotypes. Each
morphotype was mounted in PVLG and Meltzer's/PVLG (Koske and Tessier,
1983) and identi®ed based on subcellular characteristics.
Fifty uniform (0.10 m tall), two-month old, non-mycorrhizal `Volkamer' lemon
seedlings were potted into 8 l plastic pots ®lled with a pasteurized potting mixture
of coarse sand (particle size < 4.25 mm) blended with soil (3 : 1 by volume)
which had been passed through a 2 mm sieve. The soil was a Gilman clay loam
collected at a depth of 0.1±0.3 m in the A horizon from the Arizona State
130
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
University Horticultural Resource Unit, Tempe, AZ, USA. Soil analysis was as
follows: pH 7.3, EC 0.25 dS/m, 11.26 mg sodium bicarbonate extractable P/g
soil, and 13.2 g organic matter per kilogram soil. During the study all plants were
grown in a temperature-controlled glasshouse under natural irradiance (air
temp 308C day/218C night, mean maximum PAR 1200 mmol/m2/s, VPD
range 1 to 2 KPa).
At potting, inoculum from one of the ®ve AM fungal populations was placed
subjacent to the transplants. Inoculum potential was previously determined using
the most probable number method (Alexander, 1982), and each pot received
approximately 1.5 106 AM fungal propagules. All plants were fertilized with
12 g of 20N±0P±16.6 K±2Fe±1.4 Mn (slow release, N source as isobutylidene
diurea) and 22 g of 0N±19.6P±0K±12S (concentrated superphosphate).
After a one-week acclimatization period, half of the plants were randomly
assigned to either a continually moist or periodically dry watering treatment.
Over a four-month period, plants subjected to the continually moist treatment
were watered in excess of container capacity when soil matric potential (water
tension) decreased to ÿ0.01 MPa (approximately a 3-day cycle). Plants subjected
to the periodically dry treatment were watered in excess of container capacity
when potting soil water tensions fell to ÿ0.06 MPa (approximately a 12-day
cycle). Soil water tension was monitored with tensiometers (Soilmoisture
Equipments, Santa Barbara, CA, USA) inserted into the soil so that the ceramic
tips were located approximately one-half the distance between the surface and the
container bottom and 4 cm from the container wall.
Approximately three months after the initiation of watering frequency
treatments, leaf gas exchange and water potential measurements and soil
respiration measurements were made on the second day after an irrigation event
when soil water tension and plant water status were nearly uniform in all
treatments. Leaf gas exchange measurements were made at mid-day on recently
expanded leaves of similar appearance (approximately the seventh leaf from the
stem apex) on each plant using an infra red gas analyzer (IRGA, LI-COR 6200;
LI-COR, Lincoln, NE, USA) and 0.25 l chamber attachment. A pressure bomb
(Soil Moisture Equipments, Santa Barbara, CA, USA) was used to measure predawn and mid-day leaf water potential. Each water potential measurement was
based on a single leaf per plant using recently fully expanded leaves of similar
appearance (approximately the eighth leaf from the apex). For soil respiration
measurements, polyvinyl chloride cylindrical collars (7.0 cm height, 10.1 cm
inside diameter) were inserted into the potting soil to a depth of about 2 cm one
day before the measurements were made. A soil respiration chamber (LI-COR
6000-09) attached to the IRGA was ®tted to the collar such that the chamber air
supply manifold was 1±2 cm above the potting soil surface. A foam gasket was
placed between the collar and the chamber to prevent air leaks. Soil respiration
per pot was derived from the average of four consecutive CO2 ¯ux measurements.
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
131
At the end of the study, total stem length was measured and all plants were
harvested. Leaves and stems were separated from roots. Roots were washed free
of soil and fresh root weights were recorded. Canopy leaf area and root lengths
were measured using a digital camera interfaced with a computer (AgVision
Digital Imaging System, Pullman, WA, USA). All roots were inspected optically
(with the aid of a stereomicroscope) for pathogens and symptoms of disease and
appeared to be healthy. A sample of 1 cm length root pieces (approximately 1 g
fresh weight) was collected from each tree and ®xed in a formalin±acetic acid±
alcohol solution. Roots were stained using 0.05% trypan blue in acidic glycerol
(Koske and Gemma, 1989). The magni®ed intersections method (McGonigle
et al., 1990) was used to quantify AM fungal colonization of roots. Roots and
shoots were oven dried at 658C for 48 h and dry weights were measured. An
estimation of the carbon cost to bene®t ratio (RT/AT) of plants in response to
treatments was calculated as
RT =AT RSP RDW SA= A LA;
where RSP is the speci®c soil respiration (mmol/m2/s/g root dry weight), RDW the
total root dry weight (g/plant), SA the container soil surface area (0.042 m2),
A the carbon assimilation ¯ux (mmol/m2/s), and LA is the canopy leaf area
(m2/plant).
Phosphorus concentrations in dry, pulverized leaves were determined by the
ascorbic method (Watanabe and Olson, 1965). Dry, pulverized tissue was boiled
in ethanol to remove sugars. Starch was extracted from the remaining tissue pellet
by digestion in a solution of a-amylase and amyloglucosidase. Carbohydrate
concentrations in the resulting solutions were determined using a modi®ed
anthrone method with phenol substituted for anthrone (Scott and Melvin, 1953).
The experiment was arranged in a split plot (continually moist and periodically
dry) completely randomized block design with ®ve AM fungal inoculum
treatments and ®ve replications for a total of 50 plants. All data was subjected to
the general linear model (GLM) procedure (SAS Institute, Cary, NC, USA) for
analysis of variance. Percent AM root colonization data were arcsin transformed
to approximate a normal distribution for analysis of treatment effects. Actual
percentage means were reported. Comparisons among treatments were made with
Duncan's Multiple Range Test.
3. Results
AM species composition of inoculum and mycorrhizal colonization responses.
A range of 4±7 AM fungal species were detected in the inoculum mixtures
(Table 1). The lowest number of species was detected in the Yuma orchard
inoculum while the highest number was in the Borrego Springs desert inoculum
132
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 1
Arbuscular mycorrhizal fungal species composition of Borrego Springs (BS), Padre Canyon (PC),
Verde River (VR), Mesa (ME) and Yuma (YU) inoculum
AM fungal species and authority
Acaulospora trappei Ames & Linderman
Glomus eburneum Kennedy, Stutz & Morton
Glomus etunicatum Becker & Gerdemann
Glomus intraradices Schenck & Smith
Glomus macrocarpum Tulasne & Tulasne
Glomus microaggregatum Koske, Gemma & Olexia
Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe
Glomus occultum Walker
Glomus spurcum (Pfeiffer, Walker & Bloss)
Kennedy, Stutz & Morton
Glomus versiforme (Karsten) Berch
Glomus spp. AZ112b
Relative inoculum abundance (%)a
BS
PC
VR
ME
YU
21
0
2
11
0
30
1
0
33
47
0
0
22
8
20
3
0
0
0
5
0
16
2
0
0
0
0
0
37
1
22
15
15
10
0
11
4
0
0
13
0
81
0
2
0
0
0
55
11
0
0
0
2
a
Number of AM fungal spores for each species as a percentage of the total number of spores in
a 100 cm3 sample.
b
Undescribed species number refers to accession number at International Collection of
Arbuscular and Vesicular±Arbuscular Mycorrhizal Fungi, West Virginia University, Morgantown,
WV, USA.
(Table 1). Glomus microaggregatum was the only species detected in all of the
inoculum treatments. The Yuma orchard inoculum was distinctive in that > 80%
of the total number of AM fungal spores were from a single species, Glomus
occultum.
There was no interaction effect of inoculum and watering frequency on AM
fungal colonization of plant roots. Plants exposed to periodic soil drying had less
total colonization, arbuscules, and vesicles than well-watered plants (Table 2).
There were no differences in total colonization between inoculum treatments;
however, arbuscular colonization was greatest in plants treated with AM fungi
from the Yuma orchard. Plants treated with the Mesa orchard or Verde River
desert inoculum had a similar number of vesicles and more than plants grown
with other inocula. No septate hyphae were observed in the roots, nor was any
necrosis of cortical cells observed.
Plant growth responses and tissue P levels. Plant growth was not affected by
the interaction of watering frequency and inoculum treatments, thus, we present
plant growth responses to watering and mycorrhizal treatments separately. At
harvest, plants grown in periodically dry soil had signi®cantly less total stem
length and shoot dry weight, lower shoot to root dry weight ratio (S/R), less
canopy leaf area, and a lower canopy leaf area to root length ratio (LA/RL) than
plants grown in continually moist soil (Table 3). Root dry weight, total root
133
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 2
Effect of watering frequency and inoculum treatments on percent root length colonized by AM
fungi
Treatment
Arbuscules
Vesicles
Hyphae only
Total
Watering
Continually moist
Periodically dry
31aab
23 b
15 a
3b
19 a
16 a
64 a
43 b
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
29
21
21
25
39
6b
4b
12 ab
17 a
5b
16
17
23
20
13
49
44
58
59
57
abc
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
Values are treatment means, n 25 for watering frequency treatments and n 5 for
mycorrhizal inoculum treatments.
b
Means followed by a different letter are signi®cantly different (a 0.05) according to LSD, or
c
Duncan's Multiple Range Test.
length, and leaf P concentrations were not signi®cantly affected by watering
frequency treatments.
AM fungal population treatments had similar effects on plant growth with a
few exceptions (Table 3). Plants treated with the Padre Canyon or Verde River
inoculum had greater shoot dry weight than plants treated with the Yuma orchard
inoculum. Plants treated with the Yuma orchard inoculum had lower total root
Table 3
Effect of irrigation frequency and inoculum treatments on total stem length, shoot and root dry
weights, shoot to root dry weight ratio (S/R), canopy leaf area, total root length, canopy leaf area to
total root length ratio (LA/RL) and leaf phosphorus concentration (P) of Citrus volkameriana
Treatment
Shoot dry Root dry S/R
Stem
weight
weight
length
(g/plant) (g/plant) (g/plant)
Canopy
leaf
area
LA/RL
Root
length
(m/plant)
P
Watering
Continually moist 0.70aab
Periodically dry 0.60 b
5.9 a
4.1 b
6.1 a
5.4 a
0.96 a
0.77 b
1.02 a
0.73 b
2.88 a
2.74 a
0.39 a
0.28 b
2.1 a
2.1 a
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
5.6 a
5.3 ab
5 .4 ab
5.7 a
3.3 b
6.0
6.3
6.4
6.4
3.6
0.93
0.84
0.84
0.84
0.91
0.86
0.87
0.91
1.12
0.63
2.8
3.3
3.1
3.0
1.7
0.30
0.26
0.29
0.37
0.37
2.1
1.8
1.8
2.3
2.7
a
0.59
0.66
0.71
0.77
0.51
ab
a
a
a
b
a
a
a
a
b
a
a
a
a
a
ab
ab
ab
a
b
a
a
a
a
b
ab
b
ab
a
a
b
b
b
ab
a
Values are treatment means, n 5.
Within treatments, column means followed by a different letter are signi®cantly different
according to Duncan's Multiple Range Test (a 0.05).
b
134
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
length and root dry weight than plants treated with the other inoculum treatments.
Plants treated with the Yuma orchard inoculum also had higher leaf P
concentrations than plants grown with the other inoculum treatments. Plants
treated with the Mesa or Yuma inoculum differed in canopy leaf area, with the
former having signi®cantly greater area than the latter. However, plants treated
with the orchard inocula had higher LA/RL than plants treated with the desert
inoculum from Padre Canyon. Mycorrhizal inoculum treatments had no effect on
S/R.
Physiological responses and carbohydrate analysis. There was a signi®cant
interaction between watering frequency and AM fungal inoculum treatments on
leaf gas exchange (Table 4). When grown in moist soil, plants treated with the
Padre Canyon desert inoculum had the highest leaf carbon assimilation (A) and
stomatal conductance (gs). In contrast, plants treated with the Yuma orchard
inoculum had the lowest A and gs, while plants treated with the Yuma orchard and
Verde River desert inoculum had the lowest water use ef®ciency (WUE)
compared with plants treated with other inocula. Mycorrhizal inoculum
treatments had no effect on CI : CA.
When grown in periodically dry soil, plants treated with Yuma orchard
inoculum had the lowest A and WUE, and the highest CI : CA (Table 4). Plants
treated with Borrego Springs or Padre Canyon desert inocula had the highest gs,
while plants inoculated with the Verde River or Mesa inocula had the lowest
CI : CA compared with plants treated with the other mycorrhizal inoculum
treatments.
Speci®c soil respiration (RSP) was affected by an interaction of watering
frequency and inoculum treatments (Table 4). Plants treated with the Yuma or
Mesa orchard inocula had greater RSP than other plants treated with the desert
inocula (P 0.02) when grown in continually moist soil. However, only plants
treated with the Yuma orchard inoculum had greater RSP that was between two
and three times more than plants treated with the any of the other inoculum
treatments when grown in periodically dry soil. Our estimated carbon cost to
bene®t ratio of plants treated with the Yuma orchard inoculum was about 1.7
(continually moist) or 2.4 times (periodically dry) higher than for plants treated
with the other inocula.
Leaf sugar levels were not affected by watering frequency, but leaf starch levels
were lower for plants in periodically dry soil compared with plants grown in
continually moist soil (Table 5). Plants grown in continually moist soil had higher
root sugar and starch concentrations than plants grown in periodically dry soil.
Leaf carbohydrate concentrations were not affected by AM fungal population
treatment (Table 5). Plants treated with the Borrego Springs desert inoculum had
higher levels of root sugar than plants treated with Verde River desert inoculum,
and higher levels of starch than plants treated with Verde River desert or Mesa
orchard inocula (Table 5). Watering frequency and AM fungal inoculum
135
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 4
Effect of watering frequency and inoculum treatments on leaf carbon assimilation (A), stomatal
conductance (gs), ratio of internal to ambient CO2 concentration (CI : CA), instantaneous water use
ef®ciency (WUE A/instantaneous leaf transpiration), speci®c soil respiration (RSP soil
respiration per gram of root dry weight), and the estimated carbon cost to bene®t ratio (RT/AT)
of Citrus volkameriana
A
(mmol/m2/s)
gs
(mmol/m2/s)
CI : CA
WUE
RSP
(mmol/m2/sg)
RT/AT
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
9.8adcb
11.6 a
10.3 bc
11.0 ab
8.7 d
108.4 bc
141.5 a
119.0 b
124.8 ab
97.5 c
0.54
0.58
0.57
0.54
0.55
a
a
a
a
a
4.1
4.2
3.8
4.2
3.6
a
a
b
a
b
0.16
0.12
0.12
0.23
0.23
b
b
b
a
a
0.045b
0.030b
0.032b
0.042b
0.064a
Periodically dry
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
10.5 a
10.0 a
10.4 a
10.5 a
8.7 b
114.9
113.0
100.8
100.5
104.2
0.54
0.56
0.50
0.49
0.59
b
ab
c
c
a
4.2
4.0
4.5
4.5
3.4
a
b
a
a
c
0.14
0.13
0.11
0.16
0.35
b
b
b
b
a
0.041b
0.043b
0.033b
0.046b
0.100a
NSc
***
*
***
***
***
Watering
Continually moist
Significance
Watering
Inoculum
Watering x
Inoculum
a
a
b
b
ab
NS
**
*
NS
***
**
NS
***
NS
NS
***
NS
a
Values are treatment means, n 5.
Within watering frequency treatments, column means followed by a different letter are
signi®cantly different according to Duncan's Multiple Range Test (a 0.05).
c
Non-signi®cant, or signi®cant at P 0.05(*), 0.01 (**), or 0.001, respectively.
b
treatments did not affect pre-dawn or mid-day leaf water potentials when
measurements were taken under similar (>ÿ0.01 MPa) soil water tensions (data
not shown).
4. Discussion
These data show that communities of AM fungi can mitigate substantially
different effects on growth `Volkamer' lemon plants. The mycorrhizal effects we
measured on plant growth may be the result of changes in photosynthate
production and/or partitioning (Syvertsen and Graham, 1990). Plants treated with
136
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Table 5
Effect of watering frequency and inoculum treatments on the concentration (% tissue dry weight) of
sugar and starch in Citrus volkameriana leaves and roots
Treatment
Leaf
Root
Sugar
Starch
Sugar
Starch
Watering
Continually moist
Periodically dry
4.88aab
4.37 a
3.10 a
2.16 b
2.33 a
1.65 b
1.41 a
0.97 b
Inoculum
Borrego Springs
Padre Canyon
Verde River
Mesa
Yuma
4.56
4.55
5.20
4.55
4.23
2.67
2.36
2.56
2.72
2.87
2.26
2.01
1.76
1.95
1.98
1.82
1.29
0.89
0.81
1.13
ac
a
a
a
a
a
a
a
a
a
a
ab
b
ab
ab
a
ab
b
b
ab
a
Values are treatment means, n 25 for watering frequency treatments and n 5 for
mycorrhizal inoculum treatments.
b
Means followed by a different letter are signi®cantly different according to LSD, or
c
Duncan's Multiple Range Test (a 0.05).
the Yuma orchard inoculum had root systems that were 43±49% smaller than
other plants, and when averaged across both irrigation treatments also had A
¯uxes that were 18% lower, and RSP and estimated carbon cost to bene®t ratio
values that were at least 2.0 times higher than plants treated with the other
inocula. These data support previous ®ndings that some AM fungi can reduce
growth of citrus if the high carbon costs of AM roots are not offset by increased
carbon assimilation (Graham et al., 1996).
Arid soils have high AM fungal diversity (Stutz and Morton, 1996), but
intensive ®eld cultivation might select for species or isolates of AM fungi that
tolerate horticultural practices, but which are not particularly bene®cial to crop
plants in terms of increased plant growth (Kurle and P¯eger, 1994). Unlike the
other AM fungal populations we evaluated, we found that the AM fungal
population from the Yuma orchard site was dominated (based on the proportion of
spores) by a single AM fungal species, G. occultum. Johnson et al. (1992) found
that G. occultum proliferated in corn ®elds, and that spore abundance of G.
occultum correlated negatively with corn plant dry mass and yield. Graham et al.
(1996) also found that aggressive strains of AM fungi enhanced plant P uptake
and growth at low soil P, but caused growth depression of citrus at high soil P
(Graham et al., 1996). Furthermore, Modjo and Hendrix (1986) linked growth
depression of tobacco plants to mycorrhizal colonization under conditions of high
soil P.
The Yuma orchard soil was very low in organic matter and available P which
might have favored proliferation of aggressive strains of AM fungi that enhance P
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
137
uptake. Though possibly bene®cial in situ at the Yuma site, G. occultum, might
have been responsible for the poor growth performance of the P suf®cient lemon
plants treated with the Yuma orchard inoculum in our study. In support of this
hypothesis, we note that plants inoculated with AM fungi from the Yuma orchard
soil had higher leaf P concentrations and more arbuscules (sites of carbon and P
exchange) per root length than plants treated AM fungi from the other soils. This
suggests that increased AM fungal activity might have resulted in higher carbon
costs to the plant.
Arbuscular mycorrhizal fungi can induce high rates of root/soil respiration
which represent considerable carbon costs to the plant (Peng et al., 1993;
Graham et al., 1996). If the carbon sink of AM roots is not compensated for by
increased A, then growth depression will result (Graham et al., 1996). High
values of RSP might indicate that the carbon cost to bene®t ratio of the AM
association to the plant is great and/or that fungal activity is high (Eissenstat
et al., 1993; Graham et al., 1996). Thus, we conclude that the Yuma orchard
inoculum presented lemon plants with the highest carbon cost to bene®t ratio,
which might explain the reduced root length and lower root dry weights found
in those plants.
Plants treated with Yuma orchard inoculum and grown under periodically dry
soil conditions had lower A and gs than other plants, but had CI : CA that was
similar or higher than other plants. Since CI did not decrease with gs, A limitation
for these plants was at least partly stomatal (Mott, 1988). Leaf P concentrations
can also limit A (Syvertsen and Lloyd, 1994), but plants treated with Yuma
inoculum had the highest leaf P which is evidence that reduced A was not caused
by P limitation. Though leaf P concentrations for all plants were suf®cient for
normal plant growth, we did not measure levels of other nutrients which might
have limited A. Morphological differences such as increased speci®c leaf weight
or reduced root hydraulic conductance could reduce A in citrus. However, speci®c
leaf weight (data not shown) was not affected by inoculum and though root
conductance was not measured, speci®c root weight (data not shown), and
root : shoot ratio were similar among inoculum treatments, suggesting that root
hydraulic conductivity might be similar among treatments.
Although plants treated with the Yuma orchard inoculum had the highest
estimated carbon cost to bene®t ratio, they did not have signi®cantly different
levels of non-structural carbohydrates from plants treated with other inocula. In
plants with adequate P nutrition, AM fungi affect root carbohydrates more than
leaf carbohydrates (Nemec and Guy, 1982), as observed in this study. Reducing
sugars are apparently the most responsive to AM infection, but are the smallest
carbohydrate fraction, so differences in sugar and starch levels caused by AM
fungi can be subtle (Nemec and Guy, 1982; Graham et al., 1997). Since only total
sugars were measured in this study, potential small differences in reducing sugars
due to AM treatment were not detectable.
138
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
Shoot growth was positively correlated to watering frequency. Plants grown
under the periodically dry soil had lower leaf carbohydrate concentrations than
plants grown under continually moist conditions. Large citrus seedlings have
greater carbohydrate concentrations than small seedlings (Nemec and Guy, 1982),
so an analysis of variance with shoot size as a covariate was done and showed that
shoot carbohydrates were not different if size was taken into account. Thus, shoot
growth was probably not limited by leaf carbohydrate concentrations in the dry
treatment, but by some other mechanism.
Shoot growth at low soil water potential can be reduced by changes in the
elastic properties of cell walls (Nonami and Boyer, 1990) or by production of
abscisic acid in the roots (Saab et al., 1990). None of the inocula showed
signi®cant effects in terms of enhancement of leaf water potential or
improvement of shoot growth. However, AM fungal inoculum did affect aspects
of plant morphology that might enhance plant water status, such as lowered leaf
area : root length or root : shoot ratios.
Root growth was not affected by soil drying, but root growth can be less
sensitive than shoot growth to soil drying (Hsiao and Jing, 1987). Lower root
carbohydrate concentrations in plants grown under periodically dry conditions
might have been a result of lower carbon acquisition in these plants since canopy
leaf area and LA/RL was signi®cantly reduced in plants exposed to periodic soil
drying.
5. Conclusion
Growth suppression of plants treated with AM fungi from the Yuma orchard,
compared with plants treated with inocula from desert soils or the Mesa orchard,
was substantial and greater in magnitude than the main effects of the watering
frequency treatments. Previous studies have reported dramatic differences in
citrus growth as a function of different mycorrhizal isolates. The causes of
differential growth can be phosphorus limitation (Graham et al., 1982; Camprubi
and Calvet, 1996) or high carbon costs (Graham et al., 1997; Johnson et al., 1997;
Graham and Eissenstat, 1998). Since P limitation was not a factor in our study,
the differences in carbon costs of the populations used probably caused the
growth effects observed. Further research is needed to determine which AM
fungal isolate or combinations of isolates are responsible for plant growth
depression. Single species from the Yuma inoculum could be isolated, and
inoculation of citrus with isolates individually and in combination would con®rm
which fungi were responsible for plant growth suppression. Analysis of the
effects of the Yuma population compared with the other inocula on growth of
citrus under P limited conditions might provide insight into management
strategies to enhance AM fungal bene®ts in citrus orchards.
M.W. Fidelibus et al. / Scientia Horticulturae 84 (2000) 127±140
139
Acknowledgements
The authors wish to thank the Arizona Citrus Research Council for partial
support of this work.
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