Influence of Humic Acid and Mycorrhiza Fungi on Some Characters of “Speedygreen” Perennial Ryegrass (Lolium perenne L.)

Abstract

This experiment was conducted to investigate the effect of humic acid and mycorrhizal fungi on chlorophyll changes, visual quality, and some characters of roots of “Speedygreen” perennial ryegrass (Lolium perenne L.) which grown in greenhouse of Tehran University from 2th Jun 6th Aug 2009. After soil sterilization, the inoculums of mycorrhizal fungi including Glomus mosseae and G. intraradices were added to pots, and then seeds were cultivated. By establishing of plants, humic acid at different levels (0, 100, 400, and 1000 mg/L) were sprayed on leaves. Nine weeks after starting treatments, the characteristics such as chlorophyll content, visual quality and root factors were measured. The result showed that humic acid had no effect on root mass, visual quality, and colonization in roots, but was effective on a, b, and total chlorophyll content, root length, fresh weight, and dry weight. Fungi mycorrhizal were effective on all measured characteristics. The effect of G. mosseae was significant on root factors, while had no effect on plant shoots. Both of mycorrhizal fungi showed the same colonization. Probably, fungi mycorrhizal might show hormone-like effects and enhance their hyphal density in soil which is depended on type of fungi species.

Full text

AgronomyJournal • Volume106,Issue2 • 2014 585
Soil Fertility & Crop Nutrition
PerennialRyegrassGrowthResponsestoMycorrhizalInfection
andHumicAcidTreatments
AliNikbakht,MohammadPessarakli,*NaghmehDaneshvar-Hakimi-Maibodi,andMohsenKa
Published in Agron. J. 106:585–595 (2014)
doi:10.2134/agronj2013.0275
Copyright © 2014 by the American Society of Agronomy, 5585 Guilford
Road, Madison, WI 53711. All rights reserved. No part of this periodical
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording , or any information storage and
retrieval system, without permission in writing from the publisher.
ABSTRACT
Arbuscular mycorrhizal (AM) symbiosis confers numerous bene ts to host plants, including improved plant growth and
nutrient acquisition e ciency. An experiment was conducted to evaluate the e ect of AM fungi (Glomus intraradices and G.
mosseae) in the presence of humic acid (HA) spray treatments (0, 100, 400, and 1000 mgL–1) on nutrient (N, P, K, Fe, and Zn)
uptake, visual quality and chlorophyll content, root growth and architecture, and colonization of perennial ryegrass (Lolium
perenne L.) Speedygreen mixture.  e results revealed that HA did not a ect plant growth signi cantly; instead arbuscular
mycorrhiza l fungal (AMF) colonization improved dry and fresh weig hts. Mycorrhizal inoculations signi cantly increased visual
quality (13 and 15% in inoculated plants compared to non-inoculated ones) that might be at least partly due to elevated total
chlorophyll content.  e AM inoculation and HA treatment resulted in improved root architecture rather than root biomass
production. Neither HA treatments nor mycorrhizal inoculation a ected N and Fe contents of the leaves; however P, K, and Zn
concentrations improved by AM inoculation. More roots were colonized by G. intraradices than by G. mosseae.  ese results
suggest that AM inoculation is bene cial in enhancing uptake of some nutrients and root development of ryegrass possibly
leading to less fertilizer input and more drought resistance.
A. Nikbakht, Dep. of Horticulture, College of Agric ulture, Isfahan Univ.
of Technology, 8415683111 Isfahan, Iran; M. Pessarak li, School of Plant
Sciences, C ollege of Agriculture and Life Sciences,  e Univ. of Arizona ,
Tucson, AZ 85721; N. Daneshvar-Hak imi-Maibodi, a nd M. Ka , Dep. of
Horticulture, Col lege of Agricu ltural S cience and Engineering, Campus of
Agric ulture and Natura l Resources, Univ. of Tehran, Karaj, Iran. R eceived 8
June 2013. *Corresponding author (pessa rak@ag.ar izona.edu or pessa rak@
email.arizona.edu).
Abbreviations: AM, arb uscular mycorrhi za; AMF, arbus cular mycorrhiz al fungi ,
mycorrhiza f ungi; Chl, ch lorophyll; HA, hu mic acid; HS, humi c substances.
Undernaturalconditions,about90%of all plant
species form a symbiotic association with mycorrhizal fungi
(AMF) (Smith and Read, 1997; Brundrett, 2002). Arbuscular
mycorrhizal symbiosis provides many bene ts to host plants,
including enhanced plant growth (Kim et al., 2010), mineral
nutrition (George, 2000), resistance to abiotic stresses, includ-
ing drought (Auge, 2001, Abbaspour et al., 2012) and salinity
(Shari et al., 2007) when compared to similar noncolonized
(Non-AMF) plants. Most grasses form an AM symbiosis
(Newman and Reddel, 1987). However, very few studies are
available on AM symbiosis of turfgrass species. Also, turfgrass
is o en grown under improved maintenance conditions. It
has been hypothesized that these plants are less dependent on
mycorrhizae and AM symbiosis and that such symbiosis would
bene t them less than other species (Pelletier and Dionne,
2004). Butler and Hunter (2008a) reported that application
of seaweed extracts and microbial inoculants treatments into
the root zone of creeping bentgrass (Agrostis stolonifera L .)
signi cantly alleviated stress tolerance. Pelletier and Dionne
(2004) showed that a lawn mixture of Kentucky bluegrass (Poa
pratensis L.), red fescue (Festuca rubra L.), and perennial rye-
grass (Lolium perenne L.) inoculated with Glomus intraradices
established more quickly than Non-AMF turfgrasses. Similar
results were shown when the grass was inoculated with G. etu-
nicatum. Our preliminary results also showed that perennial
ryegrass was successfully colonized by mycorrhizal species and
a ected by the AM symbiosis (Ka et al., 2013).
However, many commercial products consisting of humic
substances (HS), including humic acid (HA) and fulvic acid
(FA) have been recommended for use on turfgrasses (Liu
and Cooper, 2000). Humic acid is the main fraction of HS
and the most active component of soil and compost organic
matter (Ferrara et al., 2007).  ese compounds have many
bene ts such as phyto-hormone-like activity (Pizzeghello et
al., 2001; Fike et al., 2001) that directly and indirectly have
stimulating e ects on the physiological processes of plant
growth (Yang et al., 2004). Several researchers have noted
that foliar application of HA positively a ects plant growth.
In this respect, there are e ects on uptake of nutrients (Adani
et al., 1998; Tejada and Gonzalez. 2003), photosynthesis (Liu
et al., 1998), increasing root growth (David et al., 1994) and
enhancing seed germination and seedling growth (Dorer and
Peacock, 1997).  e use of HA has o en been proposed as a
method to improve crop production (Nikbakht et al., 2008).
However, these materials have become the most commonly
used organic materials in golf course turf management
Published March 6, 2014

586 AgronomyJournal • Volume106,Issue2 • 2014
(Clapp et al., 1998) and many researchers claim that proper
use of these products could improve turfgrass quality and
physiological growth, while reducing total mineral fertilizers
consumption (Zhang et al., 2003b). Liu et al. (1998) reported
that 400 mg L–1 commercial preparation of HA could enhance
net photosynthesis, root dehydrogenase activity and root mass
regrowth in creeping bentgrass. Zhang et al. (2003a) showed
that plant metabolic enhancers (PMEs) such as seaweed extract
and HA could reduce shipment heat injury and enhance post-
transplant rooting and quality of ta ll fescue (Festuca arundinacea
Schreb.) sod. El-Khateeb et al. (2011) found mycorrhizal inoculation
to be more eective on the growth of coojong or golden-wreath
wattle, orange wattle, blue-leafed wattle, and Port Jackson willow
(Acacia saligna Labill.) compared to HA treatments.
Application of HA increases fresh weight, dry weight,
height, visual quality, and chlorophyll content of perennial
ryegrass. Each component of the hypothesis can be tested by
average of the rates applied. However, there are few studies
concerning whether inoculation of perennial ryegrass by AM
species, G. intraradices and G. mosseae would aect various
parameters compared to the untreated control. In this study,
we intend to assess simultaneous eect of HA application and
mycorrhizal inoculation on perennial ryegrass.
MATERIALS AND METHODS
Site, Cultural Conditions, Host Plant
and Soil Preparation
e experiment was conducted in pots and in the natural
condition (open air) at the University of Tehran, Karaj, Iran
(altitude 1320 m, latitude 35°48 N, longitude 51° E) in spring
and summer of 2009. Experimental pots consisted of PVC
tubes (60 cm in length and 15 cm in diameter with holes
pierced at the bottom for drainage, Persian Pipe, Tehran,
Iran). e soil was sandy loam (79% sand, 7% clay, and 14%
silt), pH 8.3, electrical conductivity (EC) 0.55 dS m–1, total N
0.02 g kg–1, P 15.9 mg kg–1 and K 120 mg kg–1. It was sieved
and sterilized at 120°C for 2 h in an oven to eliminate native
microorganisms including soil fungi (Aono et al., 2004) and
transferred to the pots.
ere were three inoculum treatments: two VAM fungus
inoculums (G. intraradices and G. mosseae) and a non-
inoculated control. e AM fungus was provided by the
Institute of Soil and Water Research, Tehran, Iran. e AM
fungal inoculums were isolated from annual medic (Medicago
scutellata L.) cultured in pots containing a 4:1 mixture of
sterilized sand/soil, using annual medic for 10 wk. Inoculum
from the pot culture comprised of a mixture of spores (16,000
spores kg–1 for Glomus intraradices and 17,000 spores kg−1 for
G. mosseae (Klironomos et al., 1993), mycelium, sandy soil and
annual medic root fragments. e pots were inoculated with
the AM fungal inoculums. On the same day, pots were seeded
with a standard commercial lawn seed, perennial ryegrass
(Lolium perenne L.) Speedygreen mixture. It was a mixture
of three cultivars and purchased from Barenbrug Company,
Nijmegen, the Netherlands.
e seeds were sown at the rates of 25 g m–2. Both AM fungi
and seeds were uniformly sprinkled by hand over the surface
of the pots and mixed with a rake into the top 1 cm of the
soil. Pots of AMF treatments received the AMF inoculums by
adding 100 g of the inoculums, while the control pots received
no inoculum. Plants were irrigated daily until establishment
(almost 40 d aer sowing) and treatments were started aer plant
establishment. No fertilizer was added to the plants at this time.
Experimental Design and Treatments
Humic acid used in this work was prepared from leonardite
(containing : C, 61.2%; N, 3.13 g kg–1 dr y matter; and P, 2.89 g kg–1
dry matter) and purchased from a Chinese company (Dalian
Yano Agriculture Co., Liaoning, China). It was granule powder
and dissolved in water. e experiment included four HA
concentrations: 0 (control), 100, 400, and 1000 mg L–1, by adding
the commercially prepared HA to deionized water (DI), and
treatments were sprayed monthly during the 6-mo experimental
period until the leaves were completely wet. e experiment was a
factorial la id out in a randomized complete block (RCB) design with
four HA concentrations, three AM inoculums treatments and four
pots as replications for a total of 48 pots.
Plant Growth (Height, Fresh and Dry Weights)
Fresh weight was determined for shoots (clippings) in each
replication every 2 wk. Mowing was done at 3 cm height. en,
shoot samples were bagged, oven-dried at 70°C for 48 h and
dry weight was recorded. Plant height was measured bi-weekly
from the base to the tip of the leaves, aer initiation of the
treatments and 2 d before mowing of the turfgrass.
Turf Visual Quality
Turf visual quality was evaluated as the integration of shoot
density, uniformity, and color on a 1 to 9 scale, where 1 was the
worst quality and completely brown, 6 = acceptable, and 9 =
best quality according to the National Turfgrass Evaluation
Program (NTEP) procedure (Zhang et al., 2003a, 2003b). It
was evaluated bi-weekly aer starting the treatments.
Leaf Chlorophyll Content
Leaf chlorophyll content was determined bi-weekly aer
mowing the plants. Leaf material (0.1 g) was ground with a
chilled pestle and mortar in diuse light using 5 mL of 80%
acetone and the homogenate was centrifuged at 3000 × g for
2 min. Aliquots of 5 mL of 80% acetone were added to the
pellet and centrifuged until it was non-green. e supernatants
were pooled and protected from light before the estimation of
chlorophyll pigments. Absorbance of extracts was measured at
663 and 645 nm with a spectrophotometer (Shimadzu, Kyoto,
Japan) (AOAC, 2006). e content of total chlorophyll (Chl) in
leaves was determined using the formula given by Arnon (1949).
Total Chl (mg mL–1) = 0.02 02 × A645 + 0.0 0802 × A663
Plant Analysis
Mineral contents of plant shoots were determined 30
and 60 d aer starting the treatments and the averages were
reported. Plant samples were oven-dried at 70°C for 48 h and
were then ground to determine their mineral composition. e
determination of total N in the leaf samples was based on the
Kjeldahl method (Eaton et al., 1995). e extraction of K, P,
Fe, and Zn from the plant tissue material was performed by
using 1 M hydrochloric acid (HCl) aer dry ashing at 550°C

AgronomyJournal • Volume106,Issue2 • 2014 587
for 5 h. e concentrations of Fe and Zn were determined by
atomic absorption spectrometer (Shimadzu, Kyoto, Japan)
(AOAC, 2006) and K was analyzed by ame photometer
(ELE, Model PFP7, UK), while that of P was estimated by the
vanadomolybdophosphoric acid colorimetric method at 460
nm (Eaton et al., 1995). e colorimetrical determinations of
P were performed using a Shimadzu UV2401 PC (Shimadzu,
Torrance, CA) spectrophotometer.
Root Analysis
At the end of the experiment, the roots of the plants were
removed from the soil, washed carefully with tap water to
remove the soil and separated from shoot and thatch, weighed
and placed in plastic covers, and stored at 4°C until analyzed
(approximately 15 d aer exhumation). e WinRHIZO
system (Regent Instruments Inc., uebec City, QC, Canada)
was used to analyze stored root samples. Large root samples
were cut into shorter samples to reduce root volume and
overlap. Roots were scanned in gray scale color to determine
root morphology, including total root length (mm), diameter
(mm), and surface area (cm2). Scanner resolution was set at 157
dots per centimeter.
Colonization
To assess mycorrhizal colonization of roots, ve root
samples, each containing about 10 to 15 single plants, were
collected randomly in each pot 8 wk aer seeding and again
on 10 and 12 wk aer seeding. Roots were assessed for
colonization according to Phillips and Hayman (1970) with
some modications that are briey outlined here. Roots were
cleared in 10% KOH solution at 120°C for 15 min in hot-water
bath before KOH was removed from the root samples. e
roots were then washed with tap water and covered with 1%
HCl, which was poured o aer 3 min. en, roots were
stained in Trypan blue. Percentage of mycorrhizal colonization
was then assessed on 40 root intersections by the gridline
intersects method of Giovannetti and Mosse (1980).
Statistical Analysis
e experimental data were statistically analyzed by two-way
ANOVA with Statistical Analysis Systems (SAS) soware,
version 9.1. e signicance of the dierences between
treatments was estimated using the Least Signicant Dierence
(LSD) test, and a main eect or interaction was deemed signicant
at P ≤ 0.05. Finally, Graphs were drawn using Excel 2010 soware.
RESULTS
Plant Growth (Height, Fresh and Dry Weights)
Humic acid did not aect plant growth signicantly, except
that height of plants treated with 1000 mg L–1 HA was
signicantly less than that of the control plants; although plant
height decreased in comparison to control non-inoculated
plants (Table 1). e AMF inoculations positively inuenced
plants height. With no HA, G. mosseae was more eective
than G. intraradices and at 100 mg L–1 plant height was not
signicantly dierent comparing both Glomus species. In
contrast, in higher concentrations (400 and 1000 mg L–1) G.
intraradices was more eective than the other species (Fig.
1A). Both fresh and dry weights in inoculated plants tended
to increase compared to non-inoculated control plants (Fig. 1B
and 1C). Fresh weight in inoculated plants with G. intraradices
was higher when HA was applied on plants, whereas treated
Table1.Effectofhumicacid(HA)sprayandarbuscularmycorrhiza(AM)fungiinoculationonvisualquality,totalchlorophyllcontent,andsome
growthparametersofperennialryegrass.
Treatments
†Meansofsomegrowthparameters
TotalchlorophyllFreshweight Dryweight Height
Visualquality(1–9
scale)
––––––––––––––––– g ––––––––––––––––– cm gkg–1freshweight
HA‡,mgL–1
0 1.42±0.04a 0.94±0.04a 6.37±0.11a 7.57±0.14a 3.45±0.09b
100 1.47±0.03a 0.93±0.03a 6.42±0.10a 7.7±0.13a 3.81±0.13a
400 1.37±0.04a 0.92±0.03a 6.30±0.13ab 7.6±0.14a 3.78±0.12a
1000 1.36±0.05a 0.89±0.03a 5.94±0.09b 7.3±0.12a 3.59±0.10b
Inoculationtreatment
Control 1.26±0.02b 0.78±0.02b 6.67±0.07a 6.88±0.10b 3.38±0.11c
Glomus mosseae 1.46±0.04a 0.99±0.03a 6.48±0.09b 7.91±0.10a 3.67±0.09b
Glomus intraradices 1.50±0.04a 0.99±0.03a 5.62±0.09c 7.82±0.10a 3.91±0.09a
ANOVAsource
TRT
HA * ** ** * *
AMFungi ** ** ** ** *
AMFungi×HA * ** ** ** **
CV,% 15.54 21.59 8.57 10.13 8.63
*SignicantatP=0.05.
**Signica ntatP=0.01.
†Meanse parationwithcolumnbytheLe astSignicantDifferencetest(LS D)atα =0.01.Mea nsinthesamecol umnfollowedbythesamele ttersarenots tatisticallydif-
ferentatP=0.01.
‡Alltreatmen tswereappliedasfoliarspray s.Solublehumicacidwerereapplied30dafte rrsttreatment.

588 AgronomyJournal • Volume106,Issue2 • 2014
with G. mosseae fresh weight was greater in 0 mg L–1 of
HA. e same trend was observed in the case of dry weight,
although there was no dierence in 400 and 1000 mg L–1
among G. intraradices and G. mosseae (Fig. 1C).
Turf Visual Quality and Chlorophyll Content
Eect of dierent HA concentrations and AMF inoculation
on visual quality are shown in Table 1. Mycorrhizal
inoculations signicantly increased visual quality compared
with control treatment (P < 0.01). e best visual quality was
recorded in pots inoculated with G. mosseae in 0 mg L–1 of HA
(Fig. 2). Both AMF inoculations enhanced visual quality in all
HA concentrations when compared with the control, except
for G. mosseae treated with 100 mg L–1 HA spray (Fig. 2).
In the present experiment, application of HA improved total
Chl content, except for the highest level which was not aected by
HA spray (Table 1). Both inoculation treatments increased total
Chl content of the leaves. Plants inoculated by G. intraradices
Fig.1.Effectofhumicacidsprayandarbuscularmycorrhizalfungi(AMF)colonizationonthe(A)height ,(B)freshweight,and(C)dryweightof
perennialryegrass.MeansareseparatedbyLSDtestatP≤0.05.Verticalbarsrepresentst andarderrorofthemeans.

AgronomyJournal • Volume106,Issue2 • 2014 589
showed 15% increase in total Chl content (Table 1). As shown in
Fig. 3, total Chl content increased when plants were colonized
by G. intraradices and sprayed by HA, however in the highest
concentration of HA, both fungi species aected Chl content
equally. When no HA was sprayed, there was no signicant
dierence in Chl between the control and G. mossaea treatment,
whereas Chl between the HA treated and G. interaradices
treatment was signicantly greater than that of the control.
Root Characteristics and Arbuscular
Mycorrhiza Fungal Colonization
Humic acid signicantly aected diameter, length, and surface
area of the roots; however it did not inuence root fresh weight.
e lowest concentration of HA was more eective on root
characteristics than were the higher doses, where 1000 mg L–1
decreased root diameter (17%), root length (47%), and root
surface area (61%). Although root fresh weight declined when
inoculated by both A M fungi, but inoculation improved root
diameter, length, and surface area. is eect is, especially,
statistically signicant when G. mosseae is concerned. Although
HA application showed no signicant eect on roots mycorrhizal
colonization by G. intraradices and G. mosseae, but in general more
roots were colonized by G. intraradices than G. mosseae ( Table 2).
Nutrient Content
Neither HA treatments nor mycorrhizal inoculation
aected N content of the leaves. Phosphorus content showed
the same trend in response to HA treatments; however both
mycorrhiza species improved P accumulation in leaves (Table
3). Potassium content was signicantly enhanced by both
Fig.2.Effectofhumicacidsprayandarbuscularmycorrhizalfungi(AMF )colonizationonthevisualqualityofperennialryegrassleaves.Meansare
separatedbyLSDtestatP≤0.05.Verticalbarsrepresentstandarderroroftheme ans.
Fig.3.Totalchlorophyllcontentofperennialryegrassleavesatdifferentconcentrationsofhumicacidinthetreatmentswithnon-mycorrhizal
(control),G. mosseaeandG. intraradicesinoculation.MeansareseparatedbyLSDtestatP≤0.05.Verticalbarsrepresentst andarderrorofthemeans.

590 AgronomyJournal • Volume106,Issue2 • 2014
Table2.Effectofhumicacid(HA)spr ayandarbuscularmycorrhiza(AM)fungiinoculationonrootcolonizationandgrowthofperennialryegrass.
Treatments
Colonization
(percentofroots) Freshweight
†Meansofrootparameters
RootsurfaceareaDiameter Length
% g –––––––––––––––––––– m m –––––––––––––––––––– cm2
HA‡,mgL–1
0 37.57±1.19a 53.248±3.11a 1.37±0.034a 1813.05±117.29b 1796±59.62b
100 38.01±1.08a 56.150±3.59a 1.40±0.044a 2342.37±97.31a 2193.21±55.84a
400 38.46±1.17a 55.100±3.32a 1.41±0.047a 1977.77±79.01b 1368.74±55.23c
1000 40.63±1.40a 56.708±3.34a 1.17±0.051b 1590.58±54.11c 1356.35±86.31c
Inoculationtreatment
Control –64.34±2.69a 1.22±0.042b 1730.11±55.57b 1495.33±106.40c
Glomus mosseae 35.22±0.80b 52.37±2.70b 1.38±0.042a 2187.04±90.97a 1861.35±99.90a
Glomus intraradices 37.77±0.83a 49.18±1.40b 1.40±0.036a 1875.68±119.37b 1679.05±87.17b
ANOVAsource df
TRT 12
HA ns ns§ ** ** **
AMFungi ** ** ** ** **
AMFungi×HA ns ns ns ** ns
CV,% 10.77 17.58 10.48 10.08 9.16
**P=0.01.
†Meanse parationwithcolumnbytheLe astSignicantDifferencetest(LS D)atα=0.01.Mea nsinthesamecol umnfollowedbythesamele ttersarenots tatistica llydif -
ferentatP=0.01.
‡Alltreatmen tswereappliedasfoliarspray s.Solublehumicacidwerereapplied30dafte rrsttreatment.
§ns,nonsignicant .
Table3.Effectofhumicacid(HA)sprayandarbuscularmycorrhiza(AM)fungiinoculationonnutrientscontentofperennialryegrassleaves.
Treatments
†Meansofsomenutrientcontents
NP K Fe Zn
––––––––––––––––––––––––––––– % –––––––––––––––––––––––––––– –––––––––––––––––––mgkg–1–––––––––––––––––––
HA‡,mgL–1
0 3.80±0.091a 0.30±0.01a 1.19±0.059b 379.54±16.94a 14.39±1.09b
100 3.90±0.064a 0.31±0.011a 1.30±0.064a 375.01±15.22ab 16.11±1.33a
400 3.95±0.075a 0.30±0.12a 1.32±0.067a 349.38±16.55b 17.32±1.30a
1000 3.79±0.075a 0.30±0.01a 1.23±0.049ab 357.35±14.82ab 14.10±0.93b
Inoculationtreatment
Control 3.81±0.067a 0.24±0.008c 1.01±0.63b 400.98±10.01a 9.87±0.36c
Glomus mosseae 3.83±0.062a 0.34±0.005a 1.35±0.026a 314.70±12.69c 16.90±0.85b
Glomus intraradices 3.93±0.072a 0.32±0.0052b 1.42±0.031a 380.27±14.15b 19.67±0.90a
ANOVAsource
TRT
HA * ns§ ** ** **
AMFungi * ** ** ** **
AMFungi×HA ns ns ** ** **
CV,% 4.38 8.25 10.52 8.59 15.47
*P=0.05.
**P=0.01.
†Meanse parationwithcolumnbytheLe astSignicantDifferencetest(LS D)atα=0.01.Mea nsinthesamecol umnfollowedbythesamele ttersarenots tatistica llydif -
ferentatP=0.01.
‡Alltreatmen tswereappliedasfoliarspray s.Solublehumicacidwerereapplied30dafte rrsttreatment.
§ns,nonsignicant .

AgronomyJournal • Volume106,Issue2 • 2014 591
Fig.4.Potassium,Fe,andZncontentsofperennialryegrassleavesatdifferentconcentrationsofhumicacidinthetreatmentswithnon- mycorrhizal
(control),G. mosseaeandG. intraradicesinoculation.MeansareseparatedbyLSDtestatP≤0.05.Verticalbarsrepresentst andarderrorofthemeans.

592 AgronomyJournal • Volume106,Issue2 • 2014
HA and fungal inoculation. It was elevated by 40% in plants
inoculated by G. mosseae in comparison with non-inoculated
ones (Table 3). Higher doses of HA (400 and 1000 mg L–1)
resulted in G. mosseae activity increasing, K uptake was
greater with inoculation at HA rates of 0 or 100 mg L–1 by G.
intrardices than occurred with inoculation with G. intrardices
(Fig. 4A). Humic acid treatments were not eective on Fe
uptake and both AMF inoculations resulted in decreased Fe
uptake compared to the control. It was especially noticeable
in the case of G. mosseae in which any concentration of HA
suppressed Fe uptake by eciently inoculated plants (Fig.
4B). Zinc content increased signicantly (20%) in response
to 100 and 400 mg L–1 HA. However, comparing tissue Zn
content of the inoculated treatments at all HA levels, tissue Zn
content increased the least at the higher dose of HA (1000 mg
L–1) (Table 3). e AMF inoculation led to a greater uptake
of Zn in comparison with control plants. Zinc content rose
by 71% and was doubled in plants inoculated by G. mosseae
and G. intraradices, respectively (Table 3). e same trend was
observed in Fig. 4C where, G. intraradices was more eective
than G. mosseae in all doses of HA.
DISCUSSION
In our study, plant growth improved by AMF inoculation.
It was observed that inoculation with AM fungi resulted
in a greater growth (height, fresh and dry weights) than
control treatments, which is in agreement with the result of
Mohammad et al. (2004) and Kim et al. (2010). Charest et al.
(1997) reported similar results with G. mosseae on the growth
of two species of turfgrass and Pelletier and Dionne (2004)
showed that plants inoculated with G. intraradices had the
largest response in establishment when inoculated at seedling
period compared to inoculated plants with G. etunicatum
at seedling period. is increase in plant growth parameters
might be rst attributed to the stimulatory eect on nutrient
uptake and then the result of enhanced plant growth regulators
production in plants that have a strong stimulatory impact on
plant growth (Artursson et al., 2006). Previous works have
demonstrated that exogenous applications of HA may cause
endogenous shis in the balance of hormones, increasing
cytokinins, IAA, and ABA levels, while decreasing gibberellins
(Zhang et al., 2003a). Katkat et al. (2009) showed that HA
increased the dry weight in wheat at low concentrations.
However, species behavior may be dierent in response to HA
application. is might be the reason that foliar application
variously aects growth and development in plants. Our results
showed that foliar application of HA did not signicantly
inuence growth of perennial ryegrass. is is not in agreement
with the reports showing that plant growth increased in
response to treatments with low and medium concentrations
of HA (Chen and Aviad, 1990; Atiyeh et al., 2002). However,
it is reported that growth of some street tree species was
improved even under salinity when green waste compost with
AM fungi was used (Marosz, 2012). Our ndings suggest the
importance of further investigation to understand the impact
of humic substances, AM fungi interactions, and their eects
on the plant growth. It should be considered that a decrease
in plant height in turfgrasses, while plants are experiencing an
improvement in biomass could be of economic importance.
Any treatment controlling turfgrass height would lead to a
reduction in mowing frequency (Christians, 2007).
In the present study, mycorrhizal inoculation improvement
on the visual quality of turfgrass became well established
(Table 1). One of the major factors in a turfgrass rating
system is visual quality. Maintaining good grass color is a vital
component of turfgrass management, and the use of microbial
inoculants in turfgrass management programs may be a useful
tool to increase visual appeal, rootzone microbial activity,
and turfgrass stress tolerance (Butler and Hunter 2008b). We
did not nd HA to improve visual quality by itself. is is in
agreement with the results of some researchers showing that
no concentrations of HA could aect visual quality (Liu and
Cooper, 2000). However, there are some reports showing that
applying biostimulants such as a mixture of bacteria, FA, and
HA enhanced visual quality and color in creeping bentgrass
(Agrostis stolonifera L.) (Muller and Kussow, 2005). Similarly,
Zhang et al. (2003b) showed HA application improved turf
quality and could enhance its color, quality, and health.
e results of the present experiment highlight the role of
AM fungi and HA on increasing plant chlorophyll content
over control plants. Enhancement of chlorophyll content with
application of HS in nutrient solutions or foliar spray has been
reported by several investigators (Vaughan and Malcolm,
1995; Chen et al., 2007; Ayman et al., 2009). It was reported
that the greater growth was achieved with the concentration
of 50 to 300 mg L–1 HA and it was related to the source of
HA (peat or leonardite) (Adani et al., 1998; Chen and Aviad,
1990). It is documented that foliar spray of HS can inuence
plant growth which can be attributed partly to the enhanced
chlorophyll content in the leaves (Chen and Aviad, 1990). Our
results are somewhat in contrast to those of Chen et al. (1999)
who reported that increased Chl content of creeping bentgrass
treated by HA was due to HA-mediated maintenance of Fe and
Zn in leaves at sucient levels. Some researchers have shown
that no dierences were observed for Chl content of the turf
with any HS treatment, suggesting turf color and visual quality
are not enhanced by using HS (Van Dyke, 2008). Also, Ferrara
et al. (2007) demonstrated that applying HA increased total
Chl content in the leaves when sprayed on grape plant (Vitis
spp.). Ayman et al. (2009) showed Chl content signicantly
increased by the application of HA interacted with amino
acids. Similar research was conducted by Tejada and Gonzalez
(2003) who reported the highest Chl a and b values were
from plots receiving amino acids and HA in asparagus plant
(Asparagus ocinalis L.) and Cheng et al. (2007) showed Chl
of perennial ryegrass grown in amended soil with 5 to 100%
composted sewage sludge (CSS) was greatly improved. In the
present study, AM inoculation, especially with G. intraradices,
signicantly increased Chl content of the leaves. An increase
in Chl content due to inoculation with AM fungi has been
reported previously (Boby et al., 2008; Kim et al., 2010).
Higher total Chl content may be due to changes in the plant
metabolism (Kim et al., 2010) and might have resulted in enhanced
plant growth and biomass production (Kohler et al., 2007).
Humic acid signicantly aected root growth as far as root
length and surface are concerned, especially at the lowest
concentration (100 mg L–1). Increased rooting following HA
application was found in Kentucky bluegrass (Poa pratensis

AgronomyJournal • Volume106,Issue2 • 2014 593
L.) (Zhang et al., 2003c) and other plants (David et al.,
1994; Atiyeh et al., 2002). Liu et al. (1998) reported that
HA solution had no eect on root regrowth and actually
reduced root length at low concentration, but 400 mg L–1
HA visually produced more developed root mass. In our
experiment, the roots that received 100 mg L–1 HA were more
developed and grew more than the roots of plants received
higher concentrations. e optimal levels of humic substance
to enhance root and shoot growth vary greatly, but Chen and
Aviad (1990) documented it as 50 to 300 mg L–1. However,
some researchers showed that spraying HA on plants had no
eect on root growth. Perhaps this problem is related to no
tangent HA with roots (Cooper et al., 1998; Liu et al., 1998).
In the present experiment, HA application had little eect
on some root characteristics (colonization, fresh weight, and
diameter). As a consequence, although foliar application
of HA could improve root architecture (especially, in low
concentration), it had little eect on root growth of the
plants. e origin of HA can also be of importance on root
growth. Results of Ervin et al. (2008) experiments showed that
application of peat-originated HA increased root mass by 73%
in Kentucky bluegrass, while HA from leonardite enhanced
root growth just by 34%. Turf root inoculation was not
stimulated by HA application. Hmuic acid application showed
no signicant eect on roots mycorrhizal colonization. To the
best of our knowledge, there is no extensive study on the eect
of HA application on AM fungi inoculation. Further studies
are needed to explain these observations.
It is shown that one of the principals for adaptations to
adverse soil conditions is an improvement in root structure via
AM symbiosis (Turk et al., 2006). In the present investigation,
the results indicated inoculation of perennial ryegrass with
either of the AM fungi had a positive eect on length and
surface area of the roots. e increases in root colonization
rates show that the inoculum is able to colonize turfgrass
seedling roots. Our results showed signicant dierences
between mycorrhizal species. ese suggest that G. intraradices
can be more ecient in increasing colonization in L. perenne;
that is in agreement with results of Pelletier and Dionne
(2004) and Mohammad et al. (2004) on wheat (Triticum
aestivum L.). Such dierences among mycorrhizal species have
also been reported on the level of colonization (Zhu et al.,
2000; Sanders and Fitter, 1992) and some other parameters in
plants such as mineral acquisition (Clark et al., 1999). It seems
that mycorrhizal inoculation may help turfgrass managers
to maintain healthier turfgrass under intensive management
regimes, especially during the establishment years and adverse
environmental conditions.
We found that the N content in the leaves of L. perenne did
not dier signicantly across all HA and AMF treatments.
Although P content showed the same trend across all HA
treatments, its accumulation diered dramatically between
G. intraradices and G. intraradices inoculants. Mycorrhizal
inoculation has been shown to increase uptake of P, K, and
Zn (Chen et al., 2007; Gaur et al., 2000; Kim et al., 2010)
and some other nutrients because roots were supplemented
with the AM fungal hyphae (Dong et al., 2008; Wang et al.,
2007). Increased nutrient uptake is shown in the present study
by increased P, K, and Zn recovery in the inoculated plants
as compared to the non-inoculated controls. e inoculated
plants were able to obtain greater quantities of soil P and
probably produce more plant dry matter. With the help of
AM fungi, a host plant can obtain more nutrients and plant
resistance to undesirable condition can be enhanced (Chen et
al., 2007; Gaur et al., 2000; Kim et al., 2010).
Our results showed signicant dierences in the mycorrhizal
species for ryegrass nutrition which is in agreement with
ndings of Pelletier and Dionne (2004). It appears G.
intraradices is more ecient in promoting the uptake of some
elements into turfgrass plants than is G. mosseae. Promotion
in uptake of nutrients with the addition of HA or inoculation
with AM fungi as separate treatments had been reported by
various researchers (David et al., 1994; Adani et al., 1998;
Sharif et al., 2002; George, 2000; Turk et al., 2006).
According to Katkat et al. (2009), foliar applications of HS
had signicant eect on uptake of N, P, K, Ca, Mg, Na, Fe, Cu,
Zn, and Mn from calcareous soil, and these elements increased
the dry weight of wheat in non-limed pots. e stimulation of
ion uptake following HS treatments led some researchers to
propose that these materials may aect membrane permeability
(Katkat et al., 2009). It seems that beside the source of HA
and the nature of container medium, eciency of HA also
diers according to the plant species. Liu et al. (1998) showed
HA had no inuence on the tissue concentrations of P, K, Fe,
Mo, and Zn in creeping bentgrass, although HA increased
tissue concentrations of Mg, Mn, and S. ere was no increase
in tissue P concentration reported in turfs such as creeping
bentgrass grown in sand (Liu et al., 1998; Van Dyke 2008)
or solution (Cooper et al., 1998), when HA was applied as a
foliar treatment, however tissue P levels increased when HA
was incorporated into sand (Cooper et al., 1998). Hunter and
Andres (2004) in an attempt to control leaching of nutrients
in growing creeping bentgrass by applying HA observed that
HA did not aect the nutritional status of the leaf tissue. ey
reported that HA had an eect on root architecture, growth
of the plant and also the plant’s resistance to drought. It is
documented that HA might have limited promoting eects
when plants receive adequate nutrients (Cooper et al., 1998).
Our treatments did not elevate P content. is is in agreement
with results reported by Bidegain et al. (2000). ey believe
that HA can improve P uptake in ryegrass only in the case
of very low P availability. It is reported that ryegrass usually
shows small positive responses to AM inoculation in terms of
nutrient uptake, due to the fact that this plant species has an
extensive root system (Dong et al., 2008; Chen et al., 2007;
Pelletier and Dionne, 2004). However, in our study it seems
that plants absorbed more mineral elements due to the better-
developed root systems. Surprisingly, neither HA treatments
nor fungal inoculation improved Fe content of the leaves. Even
Fe content dropped dramatically in plants inoculated by AM
fungi. is result is in contrast with ndings of Nikolic et al.
(2003) who reported that Fe reduction in cell apoplast by HS
might be the reason why Fe was accumulated in leaf tissues.
It seems HS has been shown to maximize the ecient use of
some nutrients, reduce fertilizer costs, and help release those
plant nutrients presently bound in minerals and salts, especially
when incorporated in soil as it is shown on bean (Vicia faba
L.) (El-ghamry et al., 2009). To the best of our knowledge, no

594 AgronomyJournal • Volume106,Issue2 • 2014
similar information has yet been provided for interaction of
HA and mycorrhiza fungi on turfgrass.
CONCLUSIONS
is study demonstrated that HA did not aect perennial
ryegrass growth signicantly when applied at ratios from 100
to 1000 mg L–1, in contrast, AMF inoculation improved fresh
and dry weights and visual quality of the grass. Although
foliar application of HA and AMF inoculation could improve
root architecture, but they did little to improve root biomass.
Inoculation could be considered as a method to decrease
some fertilizers input, including P, K, and Zn in turfgrass
management, however further investigations are recommended
to study the eect of AMF inoculation when HA is
incorporated into the soil. Based on the results of the present
study, more roots were colonized by G. intraradices than G.
mosseae. It seems that perennial ryegrass has a preference for G.
intraradices over G. mosseae. Our ndings serve as a basis for
further research to understand how fungi interact with HA to
facilitate the successful application of HA and Glomus species
in turfgrass industry.
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