The Plant Growth-promoting Fungus Penicillium spp. GP15 ... - J-Stage

Journal of Oleo Science Copyright ©2014 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess13143 J. Oleo Sci. 63, (4) 391-400 (2014)

The Plant Growth-promoting Fungus Penicillium spp. GP15-1 Enhances Growth and Confers Protection against Damping-off and Anthracnose in the Cucumber Md. Motaher Hossain1, 2, Farjana Sultana1, 3, Mitsuo Miyazawa4 and Mitsuro Hyakumachi5＊ 1

United Graduate School of Agricultural Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh 3 International University of Business Agriculture and Technology, Dhaka-1236, Bangladesh 4 Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka-shi, Osaka 577-10 8502, Japan 5 Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 2

Abstract: Plant growth-promoting fungi (PGPF) have the potential to confer several benefits to plants in terms of growth and protection against pests and pathogens. In the present study, we tested whether a PGPF isolate, Penicillium spp. GP15-1 (derived from zoysiagrass rhizospheres), stimulates growth and disease resistance in the cucumber plant. The use of the barley grain inoculum GP15-1 significantly enhanced root and shoot growth and biomass of cucumber plants. A root colonization study revealed that GP15-1 was a very rapid and efficient root colonizer and was isolated in significantly higher frequencies from the upper root parts than from the middle and lower root parts during the first 14 d of seedling growth. Inoculating the cucumber seedlings with GP15-1 significantly reduced the damping-off disease caused by Rhizoctonia solani, and the disease suppression effects of GP15-1 were considerably influenced by the inoculum potential of both GP15-1 and the pathogen. Treatment with the barley grain inoculum or a cell-free filtrate of GP15-1 increased systemic resistance against leaf infection by the anthracnose pathogen Colletotrichum orbiculare, resulting in a significant decrease in lesion number and size. Molecular and phylogenetic analyses of internal transcribed spacer sequences of the genomic DNA of GP15-1 revealed that the fungal isolate is a strain of either Penicillium neoechinulatum or Penicillium viridicatum. Key words: plant growth promoting fungus, Penicillium, damping off, anthracnose, cucumber, induced resistance 1 Introduction Biological control by beneficial microorganisms offers a natural means of protection against notorious plant pathogens. Scientists have increased their efforts to take advantage of such natural plant protection and to develop strategies by which biological control can be used effectively against plant diseases. Although biological control is subject to numerous ecological limitations, it is expected to become an important part of future control strategies against many more diseases. It is now well-established that biological control can be due to many different types of interactions between plant pathogens and biological control agents （BCAs） or their products. According to the U.S. Na-

tional Research Council, biological control is“the use of natural or modified organisms, genes, or gene products, to reduce the effects of undesirable organisms and to favor desirable organisms such as crops, beneficial insects, and microorganisms”1）. In all types of biological control, pathogens are directly, or indirectly, affected by the presence and activities of BCAs or their products. Direct effects are due to a direct interaction or physical contact between the pathogen and the BCA itself or lytic enzymes, toxic compounds, and/or antibiotics produced by the BCA. In contrast, indirect effects arise from consequences that do not involve sensing or targeting a pathogen by the BCA. Instead, stimulation of the host’ s internal defense mecha-

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Correspondence to: Mitsuro Hyakumachi, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan E-mail: [email protected] Accepted December 3, 2013 (received for review August 30, 2013)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online

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M. M. Hossain, F. Sultana, M. Miyazawa et al.

nisms by BCAs or their metabolites is the most common form of indirect effect2, 3）. While a variety of beneficial microorganisms are currently being used as BCAs, numerous studies have reported the beneficial effect of root-colonizing rhizobacteria on plant growth and protection. These have been termed“plant 4） . Plant-growth growth-promoting rhizobacteria” （PGPR） promotion can result from a direct effect on plant growth, but it is mostly related to the PGPR-mediated biological control of deleterious soil microorganisms by various mechanisms, such as competition for nutrients, siderophore-mediated competition for iron, or antibiosis5）. PGPR could also reduce pathogen infections on distal plant parts by a recently discovered mechanism usually known as 6−10） . Similar to PGPR, “induced systemic resistance” （ISR） some rhizosphere fungi are able to promote plant growth upon root colonization or after treatment with their metabolites and are functionally designated as“plant-growthpromoting-fungi （PGPF） ”2, 11）. Initially, PGPF were reported as species belonging to the genera Trichoderma, Penicillium, and Phoma and sterile fungi11−14）. However, many new fungus genera with plant growth-promotion characteristics have recently been discovered, indicating that it is a widespread phenomenon among the fungal endophytes15, 16）. These PGPF allow plants to utilize decomposing organic matter through mineral solubilization2）; to produce bioactive substances, such as plant hormones17）; to increase plant fitness under abiotic stresses 16）; and to suppress plant pathogens in the rhizosphere by antagonistic mechanisms, such as the production of hydrolytic enzymes, aggressive mycoparasitism, competition for saprophytic colonization, and the induction of plant systemic resistance12, 18−20）. Studies conducted in our laboratory have revealed that zoysiagrass（ Zoysia tenuifolia Willd. Ex. Trin.）rhizospheres harbored certain PGPF, which were capable of increasing dry matter production of a variety of crop plants by several folds2, 13）. In some cases, significant growth-promoting effects of these PGPF were observed, in terms of increased plant yield over periods of 14 weeks or more20）. These zoysiagrass rhizosphere PGPF isolates also acted as BCAs when tested against the soilborne diseases of several crop plants20）. Moreover, certain isolates were good inducers of systemic resistance against a wide range of diseases when provided as barley grain inocula, mycelia, or as cellfree filtrates2, 12−14）. Considering the importance of these fungi to their host plants, we investigated the effects of a new fungal isolate, Penicillium spp. GP15-1 from the zoysiagrass rhizosphere, on cucumber plant growth and on the severity of damping-off and anthracnose diseases.

2 Materials and methods 2.1 Fungal isolates and host plants Penicillium spp. isolate GP15-1 was originally isolated from zoysiagrass（Z. tenuifolia）rhizosphere11） and maintained as part of the PGPF collection at the Laboratory of Plant Pathology, Gifu University. The pathogens Rhizoctonia solani AG4 RO2 and Colletotrichum orbiculare（＝C. lagenarium） isolate 104T from the fungal collection of the Laboratory of Plant Pathology, Gifu University, were used as the damping-off and anthracnose pathogens, respectively. Cucumber cultivar‘Gibai,’was used as the host plant throughout the study. 2.2 Preparation of the barley grain inoculum（BGI）of fungal isolates Penicillium spp. GP15-1 was grown on 2％ potato dextrose agar（PDA）plates at 25℃ for 7 d, and was prepared for inoculum production as described by Hossain et al.13） with some modifications. Briefly, 100 g of barley kernels was suspended in 90 mL of distilled water in a 500-mL Erlenmeyer flask and autoclaved at 121℃ for 20 min. Autoclaved barley grains were inoculated with 10–15 fungal discs（diameter, 5 mm）, obtained from the actively growing margin of 7-d-old Penicillium spp. GP15-1 cultured on PDA. After 10– days of incubation at 25℃ in the dark, with shaking every alternate day, completely colonized barley grains were air-dried at room temperature（25℃）, ground to 1–2-mm particle size, and stored at 4℃ until use. To prepare the R. solani inoculum, the pathogen was cultured on PDA for 4 d at 25℃ in the dark. Autoclaved barley kernels were inoculated with mycelial discs of R. solani, following the same procedure described for the preparation of BGI of GP15-1. 2.3 Preparation of the cell-free filtrate（CF）of Penicillium spp. GP15-1 Penicillium spp. GP15-1 was grown in PDA media for 7 d. Twenty mycelial discs of the GP15-1 culture were obtained from the growing margin of a colony on PDA and transferred to a 500-mL Erlenmeyer flask containing 200 mL of potato dextrose broth（PDB）. The fungi were cultured, without shaking, at room temperature for 10 d. The crude CF was separated from the mycelia and filtered through two layers of Whatman No. 2 filter paper. The filtrate was then filter-sterilized（0.22-μm Millipore filters, Millipore Products Division, Bedford, USA）and preserved at −80℃ until use. 2.4 Testing for plant growth promotion by GP15-1 Penicillium spp. GP15-1 was tested for its ability to promote growth in cucumber plants. For this experiment, an autoclaved potting medium“Starbed” （Kyodohiryo Co. Ltd, Aichi, Japan）was used. This medium is a soil-less, peat-based potting material that contains humus, rock

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J. Oleo Sci. 63, (4) 391-400 (2014)

The Plant Growth-promoting Fungus Penicillium spp. GP15-1 Enhances Growth and Confers Protection against Damping-off and Anthracnose in the Cucumber

phosphate, and composted plant materials. The autoclaved potting medium was supplemented with powdered BGI （0.5％ and 1.0％, w/w）of GP15-1, and surface-sterilized pre-germinated cucumber seeds were sown into the potting medium/inoculum mixture（one seed per pot）. An autoclaved potting medium supplemented with autoclaved barley grains was used as the control. Each treatment consisted of three replicates, with four plants in each replicate. Plants were grown in a growth chamber for 21 d at 25℃ and 12 h of light. Shoot and root lengths and fresh and dry weights were measured. 2.5 Testing for root colonization by GP15-1 Plants were grown as described above. Plants were carefully uprooted from the potting medium at 0, 7, 14, and 21 d after planting. The collected root systems were thoroughly washed and cut into approximately 1-cm-long segments21）. Root pieces were surface-sterilized with 0.25％ NaOCl for 1 min, washed with sterile distilled water, blotted to dryness, and plated on PDA supplemented with chloramphenicol（250 μg L −1）. After incubation for 2 d at 25℃, colonies of GP15-1 growing from the root segments were identified by comparing them with the original culture of the isolate. The isolation frequency of the fungus was determined by counting the number of colony-forming root segments among 100 root segments plated per replicate2）. 2.6 Testing the suppressive effect of GP15-1 against damping-off caused by Rhizoctonia solani AG-4 RO2 The BGI of GP15-1（0.5％ and 1％ w/w）and R. solani AG-4 RO2（0.5％ or 0.75％ w/w）were mixed thoroughly with the soil-less potting medium, according to the treatments. Plastic pots were filled with the potting medium and inocula mixtures. One pre-germinated cucumber seed was sown per pot, and the seedlings were grown in a growth chamber with the same conditions as previously described. After 7–10 d of growth, disease severity（DS） was measured using the disease index scale 0–4, where 0＝ no lesions on hypocotyl; 1＝＜10％ lesions on hypocotyl; 2 ＝≥ 10–50％ lesions on hypocotyl; 3＝≥ 50–90％ lesions on hypocotyl; 4 ＝ dead seedling. The degree of disease suppression attained by inoculating plants with Penicillium spp. GP15-1 was calculated on the basis of DS as follows: Protection＝［（A−B）/A］ ×100％, where A＝DS in control plants and B ＝ DS in treated plants. 2.7 Testing the suppressive effects of GP15-1 against anthracnose caused by Colletotrichum orbiculare The suppressive effects of GP15-1 against C. orbiculare were tested using two types of inocula of GP15-1: BGI and CF. 2.7.1 BGI experiment Cucumber plants were grown in pots for 21 d in the

same way as described for the plant growth-promotion experiment, using the same treatment sets. A spore suspension（105 spores mL−1）of the anthracnose pathogen C. orbiculare isolate 104T was prepared as described by Chandanie et al.22）. The challenge inoculation and subsequent disease assessment of the total number of lesions and area（mm2）of each lesion developed by C. orbiculare were performed in a similar manner to Shimizu et al. 23）. The total lesion area（TLA） of each inoculated leaf was calculated from the lesion diameter. The level of protection conferred by each treatment against C. orbiculare was calculated on the basis of TLA as follows: Protection＝［1（TLA in treated plants/TLA in control plants）］×10022）. 2.7.2 CF experiment Cucumber plants were grown in pots containing commercial Star Bed potting medium for 21 d at 25℃ in growth chambers with 12-h dark/12-h light. The induction treatment with the CF of GP15-1, the challenge inoculation with the spore suspension of C. orbiculare, and the subsequent disease assessment were carried out in a similar manner to Shimizu et al.23）. 2.8 In vitro interaction To test whether GP15-1 was antagonistic to C. oribiculare and R. solani in culture, 5-mm disks of GP15-1 and each pathogen cultured on PDA for 5 d were placed simultaneously on the same medium in petri plates, at a distance of 5 cm from each other. For determining the effect of the CF of GP15-1 on the mycelial growth of C. orbiculare, 200 μL of the CF was used to flood the surface of a PDA dish to form a thin layer. The CF was replaced with 200 μL of sterile distilled water as a control. A 5-d-old culture disk of the pathogen was placed in the center of the PDA dish containing the CF. All the plates were incubated at 25℃ for 10 d. The growth rate of each fungus was measured, and the zone of inhibition, if any was present, was also recorded. 2.9 Genomic DNA extraction The fungal isolate for DNA extraction was cultured in 200 mL of PDB at 25℃. After 3–4 d of incubation, the mycelial mat was harvested by filtration and washed with distilled water three times in a Buchner funnel. The mycelium was blotted between two Whatman No. 1 filter papers and stored at −80℃ until use. Total genomic DNA was extracted as described by Toda et al. 24）. Briefly, frozen mycelia were ground, suspended in extraction buffer（10 mM Tris-HCl pH 7.5, 100 mM EDTA, 0.5％ sodium dodecyl sulfate（SDS） , and 100 mM LiCl） and heated to 50℃ for 15 min. The supernatant obtained by centrifugation（15,000 rpm）was extracted with phenol-chloroform-isoamyl alcohol（25:24:1）. The DNA was precipitated with isopropyl alcohol, rinsed with 70％ ethanol, and re-dissolved in a 50-μL volume of TE buffer （10 mM Tris-HCl, pH 7.5, 1 mM 393

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EDTA） . After treatment with 10 μg RNase A （Sigma Chemical, USA）at 37℃ for 1 h, the DNA was extracted successively with Tris-saturated phenol, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol（24:1）, and diethyl ether. Isopropyl alcohol was added to the resulting solution, and the mixture was placed on ice for 10 min. The DNA precipitate was collected by centrifugation, washed with 70％ ethanol, and dissolved in 50 μL of TE buffer. 2.10 Sequence analysis of rDNA-ITS Polymerase chain reaction（PCR）with forward primer ITS-1（5’ -TCC GTA GGT GAA CCT GCG G-3’ ）and reverse primer ITS-4（5’ -TCC TCC GCT TAT TGA TAT GC-3’ ）was used to amplify rDNA-ITS regions of the fungal isolate. PCR amplification and sequence reaction of rDNA-ITS regions of GP15-1 were carried out according to the method described by Hayakawa et al.25）. At least two complete sequences were obtained for each ITS region. Sequences for each region were edited using Chromas Lite 2.0（http://www.technelysium.com.au/chromas.html）. The rDNA-ITS sequence of the isolate has been deposited in the GenBank database（accession nos. KC847081）. The BLAST search program http://blast.ncbi.nlm.nih.gov/Blast. cgi was used to search for nucleotide sequence homology in fungal ITS regions. The highly homologous sequences obtained were aligned by ClustalW using MEGA version 4.0 software 26）, and a neighbor-joining tree was generated using the same software. A bootstrap replication （1000 replications）was used as a statistical support for the nodes in the phylogenetic trees. 2.11 Statistical analysis The experimental design was completely randomized, consisting of three replications for each treatment. The experiment was repeated at least twice, and treatment means obtained were separated using Fisher’ s LSD test or Student’ s t-test（α＝0.05）. All analyses were performed using XLSTAT Pro statistical analysis software（Addinsoft, New York, USA） .

3 Results 3.1 Effect of Penicillium spp. GP15-1 treatment on cucumber plant growth The effect of Penicillium spp. GP15-1 treatments on cucumber shoot and root growth was studied. The treatment of potting medium with two inoculum densities of GP15-1 significantly increased the shoot fresh and dry weights of cucumber plants 3 weeks after planting. Shoot fresh and dry weights exhibited a 46％ and 176％ increase, respectively, at the 0.5％（w/w）inoculum level, and a 37％ and 100％ increase, respectively, at the 1.0％（w/w）inoculum level（Table 1） . Shoot length of the cucumber plants in GP15-1-treated pots had also increased, exhibiting a 42％ and 26％ increase at the 0.5％ and 1.0％（w/w）inoculum levels, respectively. Root biomass yield and root length responded similarly because of the addition of GP15-1 to the soil. The plants had a 36％ longer root, with 26％ and 125％ greater root fresh and dry biomass weights, respectively, at the 0.5％（w/w）inoculum level. However, root length and root fresh and dry weight increments were slightly lower at the 1％（w/w）inoculum level, exhibiting a 30％, 22％, and 75％ increase, respectively. 3.2 Root colonization ability of Penicillium spp. GP15-1 The root colonization ability of GP15-1 in cucumber plants was also examined after growing plants in GP151-treated soil. The results reveal that GP15-1 was isolated at higher frequencies during the first 7 d of seedling growth, on average 88％ and 95％ at the 0.5％ and 1.0％ inoculum concentrations, respectively（Fig. 1）. The re-isolation frequency of GP15-1 was significantly higher in the upper than in the middle and lower root parts during the first 14 d of seedling growth. However, re-isolation frequency reached 100％ in all three root segments by 21 d and did not vary between the two inoculum densities. No Penicillium was detected in the control plants. 3.3 Effect of Penicillium spp. GP15-1 treatment on the suppression of cucumber damping-off caused by Rhizoctonia solani AG-4 RO2 Growing cucumber seedlings in potting medium treated with 0.5％ and 1.0％（w/w）inoculum levels of GP15-1 significantly inhibited damping-off symptom development by

Table 1　‌Effect of inoculation with two inoculum densities (0.5 and 1.0% w/w) of Penicillium sp. GP15-1 on shoot and root growth of cucumber plants 3 weeks after planting. Treatment

Shoot Fresh weight (g) *

Root

Dry weight (g)

Length (cm)

Fresh weight (g)

Dry weight (g)

Length (cm)

Control

1.76±0.06b

0.17±0.02b

11.16±0.40b

0.92±0.05b

0.04±0.003b

12.29±0.64b

GP15-1 (0.5%)

2.57±0.07a

0.47±0.06a

15.84±0.041a

1.16±0.06a

0.09±0.003a

16.73±0.54a

GP15-1 (1%)

2.42±0.16a

0.34±0.03a

14.07±0.37a

1.12±0.03a

0.07±0.004a

15.94±0.42a

*Values are means±SEM (n = 12). Data within the same column followed by different letters are significantly different (p ≤ 0.05). 394

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Fig. 1　Isolation frequencies of plant growth promoting fungal isolate GP15-1 from the upper (toward stem) (top), middle, and lower (toward root tip) (bottom) root segments of 7-, 14-, and 21-day-old cucumber seedlings grown in potting medium amended with 0.5% (A) and 1.0% barley grain inocula. Table 2　Necrotic lesion development on the leaves and protection against anthracnose of cucumber plants treated with barley grain inoculum (0.5% and 1.0% w/w) and cell free filtrate (CF) of Penicillium sp. GP15-1 following inoculation with Colletotrichum orbiculare. Treatment

Mean Lesion number/leaf *

TLA (mm2)

% Protectiona

Control

19.53±0.19a

286.63±5.31a

GP15-1 (0.5%)

17.00±0.25b

162.57±4.47b

43.28

GP15-1 (1%)

16.47±0.26bc

119.96±3.09c

58.15

CF

16.33±0.21c

102.90±2.22d

64.10

TLA, total lesion area *Values are means±SEM (n = 12). Data within the same column followed by different letters are significantly different (p ≤ 0.05). a Protection = [1 － (TLA in treated plants/TLA in control plants)] ＊100, where TLA = sum of the area (mm2) of lesions caused by Colletotrichum orbiculare on leaves of cucumber plants. 47％ and 74％, respectively, compared to control plants inoculated with a 0.5％（w/w） inoculum of R. solani （Table 2） . When the pathogen inoculum level was increased to 0.75％（w/w）, a 35％ and 60％ reduction in damping-off severity was observed in seedlings treated with the 0.5％ and 1.0％（w/w）inocula of GP15-1, respectively, compared to pathogen-inoculated control plants. 3.4 Effect of Penicillium spp. GP15-1 treatment on the suppression of cucumber anthracnose caused by Colletotrichum orbiculare isolate 104T To investigate whether GP15-1 could protect cucumber plants against infection by C. orbiculare, plants were treated with BGI and the CF of the fungus independently. Six days after the challenge with C. orbiculare, the plants developed typical anthracnose disease symptoms, consisting of small, irregularly shaped, dark-colored lesions, and

were assessed for disease by determining the total number of lesions and total lesion area per leaf. Results showed that cucumber plants grown in soil supplemented with the BGI of GP15-1 had a significantly reduced lesion number and total lesion area compared to control plants, exhibiting 43％ and 58％ protection at the 0.5％ and 1.0％（w/w）inoculum rates, respectively（Table 3）. Treatment of cucumber leaves with the CF of GP15-1 also clearly reduced the total number of anthracnose lesions, as well as reducing lesion area, compared to non-treated control plants. Plant treated with the CF exhibited a 16％ decrease in lesion number and had a 64％ smaller lesion area of C. orbiculare infection in the challenged leaves compared to control plants （Table 3） .

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Table 3　Disease severity of damping-off in cucumber seedlings and the corresponding protection afforded by pre-inoculation with two inoculum densities (0.5% and 1.0% w/w) of Penicillium sp. GP15-1 following inoculation with two inoculum densities (0.5% and 0.75% w/w) of Rhizoctonia solani. Treatment Control

0.5% R. solani a

Disease severity

0.75% R. solani b

% Protection

*

2.27±0.27a

Disease severity

% Protection

3.20±0.17a

GP15-1(0.5%)

1.20±0.13b

*

47.06

2.07±0.12b

35.42

GP15-1(1.0%)

0.60±0.14c*

73.53

1.27±0.18c

60.41

* Values are means ± SEM (n = 12). Data within the same column followed by different letters are significantly different (p ≤ 0.05). a Disease severity was measured on a 0–4 scale in which 0 = no lesion on hypocotyl (healthy seedling); 1 = lesions cover ≤ 10% of hypocotyl; 2 = lesions cover 11–50% of hypocotyl; 3 = lesions cover 51–90% of hypocotyl; 4 = dead seedling. b Protection (%) = [(A-B)/A] × 100 in which A = disease severity in untreated control plants and B = disease severity in treated plants.

Fig. 2　Phylogenetic analysis of Penicillium sp. GP15-1. Distance tree using neighbor joining method was constructed for 11 clades (10 references and one clone) with bootstrap consensus using MEGA 5. 3.5 In vitro interaction between Penicillium spp. GP15-1 and each of the pathogens The growth of C. orbiculare and R. solani was not inhibited in a dual culture with GP15-1 on PDA, when compared with growth in the absence of PGPF. Moreover, PDA supplemented with its CF did not restrict the growth rate of C. orbiculare. 3.6 Identification of Penicillium spp. GP15-1 The complete ITS region DNA sequence（ITS-1, 5.8S, ITS-2）consisted of 585 base pairs. The sequence was submitted to NCBI GenBank, and it was given accession no. KC847081. The sequence shared 100％ nucleotide identity and 99％ quarry coverage with the ITS sequences of differ-

ent strains of P. neoechinulatum and P. viridicatum in the BLAST search, confirming that GP15-1 is a member of this group. Based on the maximum sequence homology percentage, query coverage, and the lowest E value, ITS sequence data of four isolates of P. neoechinulatum and six isolates of P. viridicatum were selected for phylogenetic analysis, while Trichoderma harzianum isolate A14 was used as an out-group. A distance tree was constructed using the neighbor-joining method, with 1000 bootstrap replications. Our results indicated that GP15-1 formed a subclade with strains of P. neoechinulatum and P. viridicatum in this tree（Fig. 2）. On the basis of sequence homology and phylogenetic analysis, isolate GP15-1 was thus identified as either a strain of P. neoechinulatum or P.

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viridicatum.

4 Discussion The use of microorganisms to promote plant growth and to control plant pests continues to be an area of rapidly expanding research. The capacity for specific root-colonizing fungi and rhizobacteria to increase the growth and yield of crop plants is currently attracting considerable attention. Specific PGPF or PGPR strains are initially selected from several hundred root-colonizing bacteria and fungi isolated from excised roots of cultivated and wild plants. Potential PGPF or PGPR are screened for their ability to inhibit the growth of various bacterial and fungal pathogens. In the present study, the growth-promoting capacity of the fungal isolate Penicillium spp. GP15-1 was established by conducting a bioassay experiment on cucumber plants. Application of the fungal isolate greatly promoted cucumber shoot length, root length, and biomass. This result indicates that the fungal isolate forms a symbiotic relationship with the host plant, leading to the enhanced growth of the host. Plant growth promotion by Penicillium spp. has been demonstrated in several studies2, 13, 15, 17）. The recent discovery of the production of large amounts of gibberellins by these fungi largely explains the mechanism involved in their growth-promoting effects on plants26）. However, stimulated root growth by GP15-1 also indicates the possible involvement of another important plant growth hormone: auxin. Contreras-Cornejo et al.27） showed that auxin plays a major role in plant growth promotion in Trichoderma virens. Khan et al.16） recently found the presence of both gibberellins and indole acetic acid（auxin）in the CF of a PGPF, Paecilomyces formosus LHL10. The other possible mechanism of growth promotion could be the ability of the fungal isolate to colonize the roots and to provide minerals to the plant in a more accessible form2）. However, the ability of PGPF to degrade barley grains and release nutrients, thus additively stimulating plant growth, cannot be ruled out. Shivanna et al.20）showed that the ammonium-N content of barley grains colonized by certain PGPF isolates was increased due to colonization by these isolates, and consequently the ammonium-N could be more readily utilized by roots, leading to an enhancement of plant growth. In our study, the greatest growth promotion effects of GP15-1 were observed when its inoculum was applied at a 0.5％ rate, rather than a 1.0％ rate. Further increments of inoculum density to 1.5％ caused no growth promotion, or even growth inhibition, in some cases（data not shown）. Many fungi isolated from soil may inhibit plant growth, and plant pathogenic fungi generally cause this inhibition. However, some fungi isolated from healthy plant roots, and not normally considered as plant pathogens, can also inhibit plant growth28）. Endophytes often increase plant

fitness to withstand biotic and abiotic stresses, while there is increasing evidence that this fitness is sometime costly, because of allocation of the limited resources of the plant to fitness-relevant processes29）. At a low inoculum density of GP15-1, there may be little or no energy cost to the plant, other than the nutrition of the root-colonizing microbes, until pathogen attack occurs. However, plants grown at higher inoculum levels of GP15-1 may be stimulated to initiate constitutive production of defense-related metabolites in the absence of a pathogen, causing a cost in energy, and a diversion of resources from growth-related processes. Therefore, the inhibition of growth that occurred at higher inoculum rates could be attributed to the higher fitness cost to plants induced by GP15-1. The rhizosphere competence of BCAs comprises effective root colonization, combined with the ability to survive and proliferate along growing plant roots over a considerable time period30）. In many respects, effective BCAs are similar to good colonizers 31）, i.e., they possess mobility, fitness, and adaptability. The root colonization study revealed that GP15-1 was a very rapid and efficient root colonizer, and was isolated in higher frequencies at both inoculum concentrations during the first 7 d of seedling growth. The results further reveal that there was a significant interaction with the region of colonization during the first 14 d of seedling growth; the colonization of roots by this isolate appeared to be higher in the upper than in the middle and lower root parts. This is similar to what was observed by Meera et al.2）, that the middle and lower root part is always less colonized than the upper, by the majority of PGPF. This is probably due to the faster growth of the roots than of the hyphae30）. Moreover, this pattern of root colonization in soil also fits with observations by Rovira et al.33）, that the main zone of root exudation is located behind the apex. However, at 21 d of growth, re-isolation frequency did not vary between regions of root, indicating that GP15-1 can colonize the entire root system. Plants obtain numerous benefits from their association with PGPF. PGPF is thought to improve plant growth through the suppression of deleterious microorganisms11）. The effect of PGPF Penicillium spp. GP15-1 on plant disease was largely unknown. Our data on damping-off disease suppression indicate that simultaneous inoculation of GP15-1 with the pathogen R. solani resulted in the suppression of disease development. This is similar to the results of Chandanie et al.34）, who demonstrated that simultaneous inoculation of Penicillium simplicissimum GP17-2 and R. solani to the cucumber rhizosphere suppressed damping-off disease development. Several reports suggest that BCAs require a period of time to colonize the host surface before they can compete with pathogens and reduce their damage35−37）. We observed very high GP15-1 colonization in the roots of 7 d-old seedlings, indicating that GP15-1 was well-established in the roots of cucumber 397

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plants at this age. The inoculum potential of the pathogen is also an important factor in the disease suppression effects of GP15-1; increasing the pathogen inoculum from 0.5％ to 0.75％ decreased the percent protection provided by GP15-1. Similarly, increasing the inoculum of GP15-1 from 0.5％ to 1.0％ resulted in a considerable increase in protection against damping-off, at both pathogen inoculum densities. The PGPF isolate tested in this study, as well as in other studies, may involve several different mechanisms in the suppression of plant disease. Although many reports have attempted to establish the importance of specific mechanisms of biocontrol to particular pathosystems, biocontrol is the final result of different mechanisms acting synergistically to achieve disease control in all natural and managed ecosystems38）. The failure to produce a zone of inhibition by GP15-1 against R. solani in dual culture indicates that the production of antibiotics or toxins might not be involved in this disease suppression. However, the interaction in the rhizosphere is very complex, and the dual culture assay is ecologically irrelevant39）. Therefore, the production of pathogen-inhibitory secondary metabolites by GP15-1 during root colonization cannot be ruled out. The other mechanisms of disease suppression by PGPF are likely to include competition with pathogens for infection sites on the root surface. Shivanna et al.20） showed that PGPF reduced the colonization ability of pathogens on roots, suggesting that mechanisms other than competition for infection sites may be involved. Therefore, another possible mechanism of disease suppression could be induced resistance in cucumber plants, elicited by GP15-1. Various investigations have demonstrated that PGPF could induce resistance in cucumber against a wide range of pathogens, when introduced as colonized barley kernel to soil, or treated with its mycelial extracts, or the CF12）. We have shown that Penicillium spp. GP15-1 protects cucumber plants against C. orbiculare when applied as a soil supplement to plants. In this system, roots were treated with GP15-1, and pathogen was inoculated on the leaves, thereby remaining spatially separated on the plant. Since no direct interactions were possible between the two populations, the suppression of disease development was probably systemic. This hypothesis is supported by the fact that the treatment of the first true leaf of a cucumber plant with a CF of GP15-1 also protects the second leaf against C. orbiculare. Thus, the protective effect of GP15-1 against C. orbiculare seems to be plant-mediated. Studies have shown that PGPF and their CFs are highly effective in reducing the severity of a number of plant diseases by the induction of systemic resistance12, 21, 40, 41）. This indicates that PGPF secrete elicitor substances in their CFs. Our previous study suggested that both the 12,000-Da fraction and the lipid fraction of PGPF CFs elicited defense responses in cucumber plants12）.

PGPF are comprised of a diverse group of agriculturally important fungi28）. The identification of these fungi is important to further our knowledge of them, and to ensure their future exploitation in agriculture. The proper identification of fungi requires the study of morphological traits, as well as genomic and proteomic data, and in particular molecular sequence data. The possible existence of different morphotypes/biotypes of fungi within a single species makes molecular sequence analysis a reliable and reproducible technique for the identification of fungal species. Among molecular sequences, the internal transcribed spacer（ITS）of the nuclear ribosomal DNA cistron is one of the most frequently used markers for phylogenetics. Of the ITS regions, ITS 1 and 2 have been employed more frequently for analysis at lower taxonomic levels. Therefore, we used sequence data from the highly variable ITS region （ITS 1, 5.8S, and 2）of GP15-1, and carried out a bootstrap test. The neighbor-joining tree with bootstrapping gave a clear identification of the fungal isolate GP15-1. Isolate GP15-1 formed a subclade with other strains of P. neoechinulatum and P. viridicatum; therefore, GP15-1 is a strain of either P. neoechinulatum or P. viridicatum. Although the sequence data are limited, it could be possible that P. neoechinulatum and P. viridicatum are the same species. Until now, there has been no information on these species of Penicillium in relation to their possible roles as plant growth promoters and biological control agents. In conclusion, our study reports the general effectiveness of the application of the PGPF, Penicillium spp. GP15-1, in stimulating plant growth and reducing the disease severity of both soilborne and airborne pathogens in cucumber. Understanding its mechanism of biological control will lead to the improved application of this isolate in agriculture. Molecular studies using Arabidopsis-based model systems are currently underway to elucidate the mechanisms that cause the disease suppression effects of GP15-1 in plants.

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