ORIGINAL RESEARCH PAPER

Penicillium sp. mitigates Fusarium-induced biotic stressin sesame plants

Ramalingam Radhakrishnan • Suk-Bok Pae •

Kang-Bo Shim • In-Youl Baek

Received: 14 November 2012 / Accepted: 4 March 2013 / Published online: 12 March 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Fusarium-infected sesame plants have sig-

nificantly higher contents of amino acids (Asp, Thr, Ser,

Asn, Glu, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His,

Try, Arg, and Pro), compared with their respective levels

in the healthy control. These higher levels of amino acids

induced by Fusarium infection were decreased when

Penicillium was co-inoculated with Fusarium. Compared

with the control, Fusarium-infected plants showed higher

contents of palmitic (8 %), stearic (8 %), oleic (7 %), and

linolenic acids (4 %), and lower contents of oil (4 %) and

linoleic acid (11 %). Co-inoculation with Penicillium

mitigated the Fusarium-induced changes in fatty acids.

The total chlorophyll content was lower in Fusarium- and

Penicillium-infected plants than in the healthy control.

The accumulation of carotenoids and c-amino butyric

acid in Fusarium-infected plants was slightly decreased

by co-inoculation with Penicillium. Sesamin and sesam-

olin contents were higher in Penicillium- and Fusarium-

infected plants than in the control.

Purpose of work: To clarify the mechanism of the

biocontrol effect of Penicillium against Fusarium by

evaluating changes in primary and secondary metab-

olite contents in sesame plants.

Keywords Amino acid � Fatty acids Fusarium �Penicillium � Sesame

Introduction

Sesame plants are affected by numerous pathogenic fungi,

the most common of which are Fusarium species (El-

Shazly et al. 1999). Phytopathogen infection can change

pigment contents and disrupt normal metabolism of

amino acids and fatty acids in plants (Whiteside et al.

2012; Xue et al. 2006; Zafari et al. 2012). Fatty acid

signaling plays an important role in plant defense against

pathogen attack. The plant hormones jasmonic acid and

salicylic acid are fatty-acid derivatives with roles in plant

responses leading to protection against pathogens (Cre-

elman and Mulpuri 2002). c-Aminobutyric acid (GABA),

a non-protein amino acid that is widely distributed in all

living organisms, is involved in the stress response and

also in carbon and nitrogen metabolism (Bouche and

Fromm 2004). Sesamin and sesamolin are unique

secondary metabolites in sesame that prevent oxidative

damage induced by pathogens (Jeng and Hou 2005).

Recently, several studies have focused on the use of

non-pathogenic organisms to prevent phytopathogen

infection. The results of such studies have suggested

that biological organisms could be an alternative to

chemical fungicides, and also provide new insights

into the development of creative management strate-

gies to suppress soil borne diseases (Choudhary 2011).

Previously, we observed the biocontrol activity of

R. Radhakrishnan � S.-B. Pae � K.-B. Shim (&) �I.-Y. Baek

Department of Functional Crop, National Institute of Crop

Science, RDA, Miryang 672-803, Republic of Korea

e-mail: shimkb@korea.kr

123

Biotechnol Lett (2013) 35:1073–1078

DOI 10.1007/s10529-013-1177-7

Penicillium against Fusarium in a dual-culture system.

In the present study, we analyzed changes in primary

and secondary metabolism in sesame plants infected

with these two fungi. The amino acid, fatty acid, and

photosynthetic pigment compositions in diseased

plants were studied by Trepanier et al. (2005) and

Zafari et al. (2012). However, there are no reports on

the involvement of amino acids, fatty acids, and

lignans in plant defenses against Fusarium infection.

The objective of this study was to determine the effect

of co-inoculation with Penicillium and Fusarium on

sesame plants, based on analyses of amino acids, fatty

acids, photosynthetic pigments, lignans, and GABA.

Materials and methods

Plant growth and fungal treatments

Penicillium sp. NICS01 (accession no: JX481980)

isolated from the rhizosphere soil of peanut plants was

cultured in potato/dextrose broth (PDB) at 28 ± 2 �C.

Sesame (Sesamum indicum L. cv. 90 days) seeds were

surface-sterilized with 0.5 % sodium hypochlorite and

washed repeatedly with sterile distilled water. Surface-

sterilized seeds were sown in pots in sterilized yard

manure that had been pretreated with Penicillium sp. The

medium consisted of peat moss (13–18 % w/v), Perlite

(7–11 % w/v), coco-peat (63–68 % w/v), Zeolite (6–8 %

w/v), NH4? (*90 mg kg-1), NO3- (*205 mg kg-1),

P2O5 (*350 mg kg-1), and K2O (*100 mg kg-1). The

control plants were sown in medium treated with sterile

water. The control and Penicillium-inoculated plants

were grown in a greenhouse under a 16-h day/8-h night

cycle at 28/20 �C and 60 % relative humidity. Fifty days

after planting, 10 ml Fusarium culture (1.9 9 104 c.f.u./

ml) was applied to control plants and Penicillium-

inoculated plants under greenhouse conditions. The

experiment had a randomized block design with five

replications, each consisting of four plants per pot. The

aerial parts of the plants were harvested 15 days after first

appearance of wilting symptoms. The collected plants

were stored at -80 �C until analyses.

Amino acids, GABA, fatty acids, photosynthetic

pigments, and lignan analyses

Samples (50 mg) were dissolved in 1 ml H2O and

centrifuged at 13,0009g at 4 �C for 30 min. The

supernatant was filtered through a nylon 66 syringe

filter (0.452 lm) and 0.1 ml was then mixed with

0.9 ml lithium citrate-loading buffer (pH 2.2). Amino

acids and GABA were determined using a Biochrom

30 amino acid analyzer on a single lithium cation-

exchange resin column (4.6 9 200 mm), using nin-

hydrin as the color reactant. Chromatography data

were analyzed using Ezchrom E software. The con-

centrations of individual amino acids and GABA were

calculated by the same procedure by comparison with

a known concentration of a standard solution contain-

ing the amino acid. The proline content was deter-

mined as described by Bates et al. (1973).

Total lipids in plant samples were extracted by the

Soxhlet method using a Buchi B-811 extraction

system (Buchi Labotechnik, AG, Flawil, Switzerland).

Fatty acid methyl esters (FAMEs) were prepared from

total lipids by acid-catalyzed transesterification as

described by Christie (2003). Lipids (1 mg) were re-

suspended in 1 ml toluene, and then 2 ml methanolic

H2SO4 (1 % v/v) was added. The reaction mixture was

heated at 100 �C for 1 h, and 3 ml H2O with 2 ml

hexane was used to extract FAMEs. Fatty acids were

analyzed as FAMEs using a capillary gas chromato-

graph equipped with a HP-FFAP capillary column

(30 m 9 0.318 mm, I.D. 25 lm film). The oven was

kept at 150 �C for 1 min, and increased to 230 �C for

1 min, then to 240 �C for 2 min. Both the injector and

detector were held at 260 �C. N2 carrier gas was at

1 ml/min; the injection volume was 1 ll, and the split

ratio was 1:50. The percentage of fatty acids was

calculated from standard values of peak areas of

C16:0, C18:0, C18:1, C18:2, and C18:3 methyl esters.

To extract photosynthetic pigments, leaf material

was ground in 80 % (v/v) acetone. Chlorophylls and

carotenoids were estimated according to the methods

of Arnon (1949) and Lichtenthaler (1987), respec-

tively. For the lignan analysis, powered sample (1 g)

was extracted with methanol for 24 h and filtered

through a nylon syringe filter (0.45 lm). To identify

lignans, the filtered sample (0.7 ml) was separated

using HPLC equipped with Lichrospher 100 RP 18

column (5 lm) at 30 �C. The mobile phase was 60 %

methanol (0.1 % trifluoroacetic acid) at 0.8 ml/min.

Lignans were detected at 361 nm. The sesamin and

sesamolin contents of Fusarium- and Penicillium-

treated and control samples were calculated by

comparisons with known concentrations of their

standards.

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Statistical analysis

The effects of the fungal treatments on primary and

secondary metabolite contents in sesame plants were

analyzed by comparison of means according to

Duncan’s multiple-range test (DMRT) at a signifi-

cance level of P \ 0.05.

Results and discussion

Biological control of phytophathogen infection in

plants is an effective and environmental friendly

method for sustainable management of soil-borne

plant diseases. In the present study, sesame plant

tissues contained detectable levels of 18 amino acids

(Asp, Thr, Ser, Asn, Glu, Gly, Ala, Val, Met, Ile, Leu,

Tyr, Phe, Lys, His, Try, Arg, and Pro; Fig. 1). Among

these, Asp, Glu, and Arg were most abundant in both

infected and uninfected plants. Mycorrhizal coloniza-

tion strongly influences amino acid uptake by plants

(Whiteside et al. 2012). In the present study, Fusarium

infection significantly increased the concentrations of

18 amino acids in sesame plants compared with their

respective concentrations in the control. However, co-

inoculation of Penicillium with Fusarium inhibited

plant damage by mitigating the increase in amino acid

levels (Fig. 1). In plants, higher concentrations of free

amino acids under stress conditions are concomitant

with symptoms of damage (Silveira et al. 2001). The

mechanisms responsible for biotic stress-mediated

regulation of individual amino acids are still poorly

understood. However, asparagine plays a role in

certain physiological changes, including the accumu-

lation of secondary metabolites under adverse condi-

tions (Jia et al. 2001). The activities of some enzymes

involved in amino acid metabolism change after

pathogen infection. For example, aspartate amino-

transferase (AspAT), which can catalyze the bidirec-

tional conversion of Asn into Gln, was expressed

differently after infection by Botrytis cinerea, Phy-

tophthora infestans, and Pseudomonas syringae (Hruz

et al. 2008). A previous study hypothesized that

glutamate consumption pathways were inhibited by

the increased activity of AspAT to maintain normal

glutamate levels. In addition, glutamate might be

converted to other stress-related amino acids such as

proline, GABA, and arginine (Brauc et al. 2011).

All samples contained lower concentrations of Gly

than Ser. But both Gly and Ser were higher in

Fusarium-infected plants than in the control (Fig. 1).

Brauc et al. (2011) reported that a decrease in Pro

content in infected plants was associated with greater

susceptibility to B. cinerea infection. In contrast,

Arabidopsis plants infected with P. syringae showed a

higher Pro content than that in uninfected controls

(Fabro et al. 2004). We also observed a significantly

elevated Pro content in Fusarium-infected sesame

plants, compared with that in the control (Fig. 1).

Quantitative and qualitative changes in fatty acids

modulate the plant response to a variety of biotic and

abiotic stresses. In sesame plants, the oil and fatty

Fig. 1 Changes in amino acid contents in sesame plants infected with Fusarium and Penicillium. Bars represent means plus standard

error (n = 3). Means followed by the same letter are not significantly different (P \ 0.05) as determined by DMRT

Biotechnol Lett (2013) 35:1073–1078 1075

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acids contents changed in response to fungal infection

(Fig. 2). Those infected with Fusarium had lower oil

and C18:2 contents, while co-inoculation with Peni-

cillium treatment alleviated these stress effects by

increasing oil and C18:2 contents (Fig. 2). Fusarium-

infected plants showed increased concentrations of

C16:0, C18:0, C18:1, and C18:3 compared with their

respective levels in the control, while co-inoculation

with Penicillium with Fusarium significantly

decreased the levels of these fatty acids (Fig. 2).

There have been few studies on changes in fatty

acid metabolism in plants under biotic stress. Different

plant species show different patterns of changes in

fatty acids in response to various stresses. For

example, the endophytic fungus Paecilomyces formo-

sus positively influenced plant growth and physiolog-

ical characters of cucumber, and infected plants

showed decreased C16:0 content (Khan et al. 2012).

Xue et al. (2006) reported increased C18:0 content in

soybean seeds infected by the seed-borne pathogen

Diaporthe phaseolorum. Fungal seed colonization was

related to the oleic (C18:1) and linoleic (C18:2) acid

contents. Higher linoleic acid and lower oleic acid

contents in soybean increased the seed colonization of

D. phaseolorum. Similarly, higher levels of oleic acid

promoted the Cercospora kikuchii association with

soybean seeds. In plant–pathogen interactions, the

ratio of oleic and linoleic acids differs depending on

the plant and the causal agent (Xue et al. 2006). Our

results showed that Penicillium co-infection increased

the levels of C18:2 and oil, compared with the levels in

plants infected with only Fusarium. The regulation of

other saturated and unsaturated fatty acids could be

due to the protective effect of Penicillium against

Fusarium infection.

In terms of photosynthetic pigments, the total

chlorophyll content was lower in plants infected with

both Fusarium and Penicillium, compared with that in

the control (Fig. 3). Similar results were reported by

Zafari et al. (2012) for Phytoplasma aurantifolia-

infected citrus leaves. It was hypothesized that the

decrease in chlorophyll content could result from

increased chlorophyllase activity in infected plants

(Bertamini et al. 2002). Compared with the carote-

noids content in the control, plants infected with

Fusarium showed higher carotenoids content, while

those co-inoculated with Fusarium and Penicillium

showed lower carotenoids content (Fig. 3). This result

differs from those of Zafari et al. (2012), who observed

decreased carotenoids content in P. aurantifoliae-

infected citrus leaves, compared with that in the

control.

Under stress conditions, plants stimulate GABA

metabolism, which prevents pathogen-induced dam-

age. Fusarium-infected plants showed increased

GABA content compared with that in controls, and

Penicillium infection ameliorated the pathogenic

effects of Fusarium by reducing GABA content

(Fig. 4). A previous report suggested that an increase

in GABA content was associated with pathogen-

induced oxidative stress in plants (Janzen et al. 2001).

Sesamin and sesamolin contents were higher in plants

Fig. 2 Oil and fatty acid

(C16:0, palmitic acid;

C18:0, stearic acid; C18:1,

oleic acid; C18:2, linoleic

acid; C18:3, linolenic acid)

contents in sesame plants

infected with Fusarium and

Penicillium. Bars represent

means plus standard error

(n = 3). Means followed by

the same letter are not

significantly different

(P \ 0.05) as determined by

DMRT

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infected with Fusarium and Penicillium than in the

control (Fig. 5). To the best of our knowledge, there

have been no reports on changes in lignans in crop

plants infected by phytopathogens. These results

suggested that the increased accumulation of some

lignans in sesame may have an antioxidant effect,

inhibiting ROS production and preventing oxidative

damage in infected plants.

Conclusion

Co-inoculation of Penicillium and Fusarium signifi-

cantly mitigated the detrimental effects of Fusarium

infection on sesame plants by regulating some primary

and secondary metabolites. Compared with healthy

plants, Fusarium-infected plants accumulated higher

concentrations of free amino acids, fatty acids, carote-

noids, GABA, and some lignans, and showed decreased

concentrations of oil and chlorophyll. Furthermore,

Penicillium treatment mitigated the Fusarium-induced

changes in amino acids, fatty acids, carotenoids, and

secondary metabolite contents in infected plants. This

investigation into changes in the levels of primary and

secondary metabolites in the plant–fungus interaction

will be useful to clarify the molecular mechanism of

defensive pathways in plants and to develop biotic

stress resistance strategies.

Acknowledgments The authors wish to thank National

Institute of Crop Science, Rural Development Administration,

Republic of Korea for financial assistance from Post doctoral

Fellowship Program (2012).

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