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Antagonistic Streptomyces selection to broad spectrum for biological control of Colletotrichum spp., causal agent of anthracnose in Chilli.

Chilli (Capsicum anuum L.) is one of the most of economic crop in Thailand; but its production had a problem by plant disease, especially anthracnose same as other countries. Anthracnose disease is a major disease of chilli in worldwide, especially, in tropical and sub-tropical areas. Damage was reported in Indonesia for 10-80% (1) and Thailand up to 80% (2). The disease is caused by at least four species of Colletotrichum; C. gloeosporioides (Penz.) Sacc., C. capsici (H.Syd.) E. Butl. & Bisby, C. acutatum Simmonds ex Simmonds, and C. coccodes (Wallr.) S. Hughes (3,4).

Selection of resistant chilli cultivar was success for some species of Colletotrichum. Chemical fungicides, such as benomyl, maneb, chlorothalonil and mancozeb were popular to manage the disease. However, some chemical, e.g. benzimidazole affected differently to species of Colletotrichum (C. acutatum was found to be moderately susceptible to this fungicide, while C. gloeosporioides was highly susceptible) (5). Chemical application normally applied biweekly or monthly which can damage the environment, and lead to development of fungicide resistance strains of Colletotrichum (6). Hence, management by biocontrol agent application can be an alternative method to save environment and human health.

Streptomyces, gram -positive bacteria in the order Actinomycetales are able to produce extracellular secretions with different properties such as antibacterial, antifungal, antiviral, nematicidal, antitumor, and enzyme inhibitory activities (7,8). Cell--free culture filtrates of S. hygroscopicus SRA 14 inhibited growth of C. gloeosporioides in orchid, apparently as hypal swelling and abnormal shapes (9). The crude extract from Streptomyces sp. SRM1 culture also showed antagonistic effects against C. musae as abnormal growth of hyphae and inhibition of spore germination (10). Extracellular chitinase and 2-1,3-glucanase produced by S. aureofaciens inhibited growth of C. gloeosporioides and treatment with the bioactive components exhibited a high protective activity against development of anthracnose disease on mango trees and increased fruit yield (11). Streptomyces cavourensis SY224 produced chitinase, 2,-1,3 glucanase, and 2Furancarboxaldehyde inhibited growth mycelium and spore germination of C. gloeosporioides infecting pepper plants (12). The solid concentrate of Streptomyces sp. A1022 reduced anthracnose by C. gloeosporioides in infections of pepper and cherry tomato (13). Previously, we found that the isolates test of Streptomyces were antagonistic to Colletotrichum capsici casual agent of anthracnose disease in chilli (14). In this study, we were to 1) screen antagonistic Streptomyces isolates for broadly control several species of Colletotrichum by testing of their potential to inhibit mycelial growth, spore germination of Colletotrichum spp. and to produce hydrolytic enzyme, and 2) to evaluate biological control potential for chilli anthracnose under greenhouse conditions.

MATERIALS AND METHODS

Pathogens

Colletotrichum. capsici 21 isolates, C. acutatum 37 isolates, C. gloeosporioides 1 isolate and C. coccodes 1 isolate were isolated from chilli fruits with anthracnose symptom collected from northeast provinces of Thailand, i.e. Khon Kaen, Mugdahan and Sakhonakon, pathogenicity test on green and red fruits and identification species of Colletotrichum followed by Sutton (15) and Sutaphun at el. (3) were used for laboratory test and C. acutatum was used for green house experiment in chilli plant.

Antagonistic Streptomyces

Seven isolates of Streptomyces spp. used in this study: PR13, PR15, PR22, PR33, PR78, PR84 and PR87 provided by Petcharat Thummabenjapone were grown on arginine glycerol mineral salt agar (AGMA) medium for 7 days for testing inhibition of mycelium growth of Colletotrichum. Their culture filtrates and micro colonies were prepared by culturing on AGMA medium for 7 days, and transferring to AGMB medium on a rotary shaker (150-160 rpm) at room temperature for 7 days. The micro colonies and culture liquid were separated by filtration through Whatman paper # 1. The micro colonies were kept in 200 mL sterile bottles at 4[degrees]C whereas the culture liquid were filtrated by using a millipore membrane filter (0.45 um) and kept in 125 mL sterile flasks at 4[degrees]C. Cell suspension of Streptomyces were prepared by homogenizing of micro colonies at the speed of 24,000 rpm for 5 min, and preserved at 4[degrees]C until future use.

Evaluation of mycelium growth inhibition by dual culture method

Colletotrichum isolates were cultured on PDA, and Streptomyces isolates were grown on AGMA, both for 7 days incubated at room temperature (28-30[degrees]C). The hyphal tips of Colletotrichum and Streptomyces colonies were bored with sterile 8-mm cork borer to produce culture discs. The assay was made on two types of media: PDA (suitable media for Colletotrichum) and AGMA (proper media for Streptomyces). Each culture disc of Colletotrichum and Streptomyces was transferred on each media at opposite direction with 30 mm apart and incubated at room temperature. In a control dish, an AGMA plug was used instead of Streptomyces disc. The experiment was arranged in completely randomized design (CRD) with four replications. The inhibition zone as distance between hyphal tips of Colletotrichum and Streptomyces colony was measured, compared with hyphal tips and AGMA plug as a control, after co-culture for 5-16 days depending on species of Colletotrichum.

Evaluation of spore germination inhibition

One isolate of each Colletotrichum species were selected and cultured on PDA at room temperature for 7-10 days depending on species. Spores were harvested by flooding the surface of culture with 5 mL of sterile distilled water and gently scraping with sterile L--shape glass rod to displace the spores. The spore suspension was passed through two layers of sterile cheesecloth. The suspensions were sampled to count spore number with haemacytometer under light microscope and adjusted concentration to 105 spore mL-1 with sterile distilled water. A 1.5 mL sterile tube were filled with 100 [micro]L of culture filtrate free from cell of Streptomyces and added with 100 [micro]L of spore suspension of Colletotrichum. The volume of 80 [micro]L combined suspension was placed on a sterile slide in moist plate and incubated at room temperature about 7-14 h (depending on species) of the fungal test, based on observations and estimations of the number of spores germinated in sterile distilled water (control). The experimental design was completely randomized with four replication.

After incubation, added one drop of lactophenol blue to stop spore germination and a total of 100 spores (germinated and non-germinated) were counted on each slide, under a light microscope. Spores with germinating tube length of at least 50% greater than the normal spore size were considered germinated (16). Percentage of spore germination inhibition was calculated according to formulation as:

Inhibition of spore germination (%) = [(No. of spores germinated in distilled water - No. of spores germinated in Treatment)/No. spores germination in distilled water] x 100

Seven isolates of antagonistic Streptomyces were selected for the test using petridish assay. The colonies of Streptomyces on AGMA for 7 days were bored with sterile 8-mm cork borer to produce culture discs and transferred to different substrates, i.e. chitinase-detection agar (CHDA)17, AGMA + 0.02% larminarin (18), and casein agar medium (19) for evaluation of chitinase, 21,3 glucanase, and protease production. In a control dish, an AGMA plug was used instead of Streptomyces disc. Each substrate media were tested for five replications in completely randomized design. The clear zone or precipitated zone around the colonies appeared after 7 days was indication of hydrolytic enzyme production, and diameter of the zones was measured. Productions of chitnase and 21,3 glucanase were showed after incubating with 0.1% congo red and 1 M NaCl, while that of collagenase was reacted when saturated with [(N[H.sub.4]).sub.2]S[O.sub.4].

Effect of antagonistic Streptomyces on chilli anthracnose under greenhouse condition

Seven treatments arranged in completely randomized design with 10 replications were as follows: (1) Streptomyces-PR22 dressed seed and from-flowering spray + C. acutatum, (2) Streptomyces-PR22 dressed seed and from seedling spray + C. acutatum, (3) StreptomycesPR87 dressed seed and from-flowering spray + C. acutatum, (4) Streptomyces-PR87 dressed seed and from-seedling spray+ C. acutatum, (5) chemical dressed seed and from-flowering spray + C. acutatum, (6) untreated + C. acutatum (disease check), and (7) untreated, uninoulated (healthy check).

Two Thailand commercial chilli varieties used in this study were bird chilli var. Prik Keenu and long cayane var. Num Kiew (Chia Tai Group). Seeds of chilli were surface disinfested with 70% ethanol for 1 min, rinsed five times with sterile distilled water, and then disinfected again with 0.5% sodium hypochlorite (Mohammed et al, 2008). Seed dressing with Streptomyces was prepared by adding 1 mL cell suspension of Streptomyces (approximately [10.sup.7] cfu [mL.sup.-1]) into 200 chilli seeds. Seed dressing with chemical was prepared by mixing 100 seeds with slurry of benomyl at the rate of 3 g benomyl per 1 kg seed. The dressed seeds were incubated at room temperature for 16 h. Treated and non treated seeds were sown in sterile peatmoss in 104-hole plastic trays.

Seedlings of Streptomyces-treated seed were divided into 2 halves. The first half was sparayed with the cell suspension ([10.sup.5] cfu [mL.sup.-1]) with 1 ml of Tween 80 at seedling stage and every week. At 45 days old the seedlings were transplanted into each 24-cm pot containing soil mixture (2:1:1,v/v/v proportion of soil: manure: black rice hull). The spray was continued until harvest. The second half of seedlings, thirty days after transplanting into the same soil mixture (at flowering stage) were sprayed with Streptomyces cell suspension + Tween 80 every week until harvest.

The 45-day-old seedlings of benomyltreated seed were transplanted into each 24-cm pot containing same soil mixture. Thirty days after transplanting, chilli plants were sprayed with benomyl (rate 6 g per 20 L) + Tween 80 every week until harvest.

Sixty days after transplanting (fruiting stage), spore suspension of C. acutatum ([10.sup.5] spores [mL.sup.-1]) was sprayed over 60 mL per plant all chilli plants except uninoculated control treatment.

The data collected at five months after transplanting consists of the number and weight of fruits per plant, and length of fruit (sampling 20 fruits per plant). Numbers of diseased fruits per plant and disease severity were recorded at one month after inoculation of C. acutatum. Percentage of anthracnose incidence was then calculated as: Anthracnose incidence (%) = (No. of diseased fruits/total No. of harvested fruits) x 100 Index of anthracnose disease modified from Phialathounheuanel et al. (20) as:

1 = No infection

2 = Necrotic lesion or water soaked lesion < 5%

3 = Necrotic lesion or water soaked lesion with few acervuli ranging 6-15%

4 = Necrotic lesion or water soaked lesion with acervuli present ranging 16-30%

5 = Necrotic lesion or water soaked lesion with acervuli present ranging 31-50%

6 = Necrotic lesion or water soaked lesion with abundant acervuli > 51%

Statistical analysis

The total data received from this study were processed for analysis of variance (ANOVA).

The treatments means comparisons were done using Duncan's multiple range test (DMRT).

RESULTS

Evaluation of mycelium growth inhibition by dual culture method

All isolates of Streptomyces could inhibit the mycelial growth of any Colletotrichum when culturing together on PDA and AGMA media (Fig. 1). The distance of inhibition zone differed significantly between isolates of Streptomyces. Streptomyces-PR22 showed the greatest inhibition of mycelium growth of 4 species of Colletotrichum (Table 1).

Evaluation of spore germination inhibition assay

Culture filtrate of Streptomyces-PR22 produced the greatest inhibition of Colletotrichum spore germination (Table 2). This isolate completely inhibited spore germination of C. capsici and C acutatum, and greatly inhibited those of C. gloeosporioides and C. coccodes (Fig.2). Great inhibition also occurred in treatments of C. acutatum with PR33, and C. coccodes with PR78 and PR87

Evaluation of hydrolytic enzymes production

All isolates of antagonistic Streptomyces produced hydrolytic enzymes such as chitinase and 21,3 glucanase as clear zone around colonies showed on CHDA and AGMA+0.02% larminarin, respectively (Fig. 3). The diameter of hydrolysis zone differed significantly between isolates of Streptomyces. For casein agar method to check protease enzyme production, it was found that Streptomyces-PR78, PR15, PR84 and PR87 created clear zone of which Streptomyces-PR78 had the greatest one (Table 3).

Effect of antagonistic Streptomyces on C. acutatum infected chili under green house condition

In brid chilli var. Prik Kheenu almost all Streptomyces treatment, except Streptomyces-PR22 dressed seed + from-seedling spray, could reduce anthracnose incidence as benomyl treatment did (Table 4). In long yard var. Num Kiew same, all applications of Streptomyces reduced anthracnose incidence similarly as chemical treatment (Table 5).

Application of Streptomyces-PR22 and PR87 or chemical had no effect on fruit number, fruit weight, and fruit length in both bird chilli (Table 4) and long yard (Table 5).

DISCUSSION

The antagonist Streptomyces-PR13, PR15, PR22, PR33, PR78, PR84 and PR87 had board spectra of antifungal activity, indicated by complete inhibition of the mycelial growth for all 4 species of tested Colletotrichum isolates on PDA and AGMA media, so these Streptomyces can produce secondary metabolite on PDA which suitable for fungal culture same as AGMA was synthetic media suitable for actinomyces culture (21) which ingredient was arginine, glycerol, phosphorus, potassium, magnesium and pH suitable for enhanced secondary metabolites production of Streptomyces (22,23). All 7 isolates of Streptomyces actively produced several hydrolytic enzymes e.g. chitinase, 2-1,3glucanase, and protease. These hydrolytic enzymes, especially chitinases and 2-1, 3-glucanases, may play an important role for inhibition of Colletotrichum mycelium growth resulting in degradation of fungal cell wall (24). Previous research showed that Streptomyces aureofaciens highly produced extracellular chitinase and 2-1,3-glucanase to inhibit growth of C. gloeosporoides of mango anthracnose11 and S. cavourensis subsp. cavourensis SY224 produced lytic enzymes such as chitinase, 2-1,3-glucanase, lipase, and protease , resulting in high growth inhibition of C. gloeosporioides infecting pepper plants (12). Streptomyces-PR22 showed the greatest inhibition of mycelium growth of 4 species of Colletotrichum while hydrolytic enzymes production was no significant difference compare with other isolates, so Streptomyces-PR22 may be secreted antifungal compound more than other isolates (7,8).

Culture filtrate from extracellular of Streptomyces-PR22, produced the greatest inhibition of spore germination of all tested Colletotrichum species while the other Streptomyces presented good results for some species only: PR33 for C. capsici, C. acutatum, and C. gloeosporioides, PR87 for C. gloeosporioides and C. coccodes, PR15, PR78 and PR84 for C. coccodes. This study found that Streptomyces-PR15, PR78, PR84 and PR87 could produce both proteolytic and gelatinolytic activity, but Streptomyces-PR22 and PR33 had no proteolytic activity, so that component in culture filtrate from them may be responsible for the inhibition of spore germination of Colletotrichum. Many report indicated hydrolytic enzyme i.e. chitinase, 2-1, 3-glucanase and protease inhibited spore germination (25,26). Palaniyandi et al. (27) showed that S. phaeopurpureus ExPro 138 produced several extracellular proteases (proteolytic and gelatinolytic activity) inhibiting spore germination, spore adhesion to polystyrene surface, and appressorium formation of C. coccodes. Streptomyces-PR22 showed board spectrum for inhibition of spore germination while creation of hydrolytic enzymes were similar other isolates. Spore of all 4 species Colletotrichum incubated with culture filtrate from Streptomyces-PR22 had similar characteristic as spore was normal structure of cell wall but inner component lysis compare with spore in sterile distilled water (Fig. 4). In addition, the researcher reported an antifungal compound inhibited spore germination of fungi such as the iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination of Penicillium roqueforti conidiospores (28).

The potential of Streptomyces-PR22 and -PR87 to control anthracnose disease in chilli plants was evaluated in a greenhouse experiment with two variety of chilli (Capsicum annuum var. Prik kheenu, and C. annuum var. Num Khew) and artificial inoculation with C. acutatum. The suspension formulation of tested Streptomyces spp. was sprayed weekly from-seedling or from-flowering and continue weekly spray until harvest compared with fungicide benomyl treatment resulted that both Streptomyces-PR22 and Streptomyces-PR87 reduced number of diseased fruits in both chilli cultivars, similar to fungicide benomyl application. This result was agreeable with a report of Lee et al. (29), Streptomyces sp. A 1022 formulated into solid concentrate were protective for pepper plants from anthracnose under greenhouse condition. The application of Streptomyces-PR22 and Streptomyces-PR87 formulations is a good biocontrol agent for management of anthracnose disease.

ACKNOWLEDGEMENTS

This work was supported by Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food Research Cluster of Khon Kaen University.

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(22.) Ives, P. R., Bushell, M. E. Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology, 1997; 143: 3573-3579.

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(24.) Adams, D.J. Fungal cell wall chitinases and glucanases. Microbiology, 2004; 150: 20292035.

(25.) Oelofse, D., Dubery, I., Berger, D.K. Exo-b-1,3-glucanase from yeast inhibits Colletotrichum lupini and Botrytis cinerea spore germination. J. Phytopathology., 2009; 157: 1-6.

(26.) Lee, E.J., Ahn, Y.J., Lee, H.S., Chung, N. Biocontrol of pepper anthracnose by a new Streptomyces sp. A1022 under greenhouse condition. J. Korean Soc Appl Biol Chem., 2012; 55 (2):447-449.

(27.) Palaniyandi, S.A., Yang, S.H., Suh, J.-W.. Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes. Journal of Applied Microbiology, 2013; 115: 207-217.

(28.) Chitarra, G.S., Breeuwer, P, Nout, M.J.R., van Aelst, A.C., Rombouts, F.M., Abee, T. An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores. J Appl Microbiol., 2003; 94(2): 159-166.

(29.) Lee, E.J., Ahn, Y.J., Lee, H.S., Chung, N. Biocontrol of pepper anthracnose by a new Streptomyces sp. A1022 under greenhouse condition. J. Korean Soc Appl Biol Chem., 2012; 55(2):447-449.

Rattikan Yutthasin [1], Petcharat Thummabenjapone [1,2] *, Anan Hiransalee [1]

[1] Plant Pathology Division, Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen--40002, Thailand.

[2] Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University, Khon Kaen--40002, Thailand.

(Received: 09 August 2015; accepted: 06 September 2015)

* To whom all correspondence should be addressed. Tel & Fax: +66-43-343114; E-mail: petsir@kku.ac.th

Caption: Fig. 1. The antagonistic Streptomyces-PR22 inhibited mycelial growth of C. capsici (a1, e1), C. acutatum (b1, f1), C. gloeosporioides (c1, g1) and C. coccodes (d1, h1) compared with mycelium growth of 4 species of Colletotrichum without Streptomyces after dual cultured on PDA (a2-d2) and AGMA (e2-h2) media

Caption: Fig. 2. Effect of culture filtrate produced from Streptomyces-PR22 on spore germination of C. capsici (a1), C. acutatum (b1), C. gloeosporioides (c1) and C. coccodes (d1) C. acutatum compared with sterile distilled water (a2-d2). Scale bar had value 20 [micro]m

Caption: Fig. 3. Production of hydrolytic enzymes by Streptomyces isolates: (a) chitinase on CHDA, (b) p 1,3 glucanase on larminarin AGMA and (c)protease on Casein agar medium
Table 1. The distance of inhibition zones between antagonistic
Streptomyces colony and hyphal tips of C. capsici, C. acutatum, C.
gloeosporioides and C. coccodes isolates after co-cultured on PDA and
AGMA media

Streptomyces     C. capsici      C. acutatum
isolate
               PDA      AGMA    PDA     AGMA

PR13           10.7cd   11.8b   11.6b   10.6c
PR15           10.7cd   11.2b   6.3d    9.5d
PR22           15.8a    16.2a   17.7a   16.3a
PR33           9.3e     9.4c    7.2c    11.3b
PR78           11.9b    11.4b   6.7cd   9.0e
PR84           9.9de    9.9c    6.3d    8.7e
PR87           11.1c    10.3c   5.4e    8.1f
Control        0.0f     0.0d    0.0f    0.0g
C.V (%)        3.35     3.84    4.52    2.12

Streptomyces    C. gloeos-       C. coccodes
isolate          porioides

               PDA      AGMA    PDA      AGMA

PR13           19.5bc   5.4c    16.0b    7.3a
PR15           18.5bc   7.3bc   13.5cd   9.8a
PR22           25.5a    16.0a   20.5a    10.3a
PR33           18.5bc   6.0bc   12.5d    3.0c
PR78           21.0b    9.3bc   14.5c    6.0ab
PR84           17.8bc   8.3bc   9.8e     7.8a
PR87           16.8c    10.3b   10.0e    7.0ab
Control        0.0d     0.0d    0.0f     0.0d
C.V (%)        9.48     12.65   4.07     16.78

Control = AGMA plugs

Means of inhibition zone from 21 isolates of C. capsici, 37 isolates
of C. acutatum and each 1 isolate of C. gloeosporioides and C.
coccodes.

Means within column followed by a common letter are not significantly
different according to DMRT (P < 0.05).

Table 2. Inhibition of spore germination of 4 species of
Colletotrichum by culture filtrate of 7 isolates of antagonistic
Streptomyces

Streptomyces            % inhibition of spore germination
isolates
                 C. capsici   C. acutatum   C. gloeos-   C. coccodes
                                             porioides

PR13              -4.2c        9.2c          -0.2d        1.6e
PR15              -91.3e       7.4cd         2.3cd        27.1c
PR22              100.0a       100.0a        88.5a        88.8a
PR33              58.2b        87.5b         12.3b        4.4e
PR78              -93.5e       -8.3ef        5.3c         78.0b
PR84              -91.9e       -16.7f        -0.2d        11.3d
PR87              -65.2de      -3.9de        11.6b        89.3a
control-AGMB      -52.2d       1.8cde        0.4d         0.9e
control-          0.0c         0.0cde        0.0d         0.0e
  distilled
  water
C.V. (%)          26.95        25.26         19.98        14.01

Means within column followed by a common letter are not significantly
different according to DMRT (P < 0.05).

Table 3. Diameter of hydrolysis zone of hydrolytic
enzymes produced by isolates of Streptomyces on
different substrates

Streptomyces   Diameter of hydrolysis zone (cm)
isolates
               CHDA    laminarin   casein

PR13           4.12a   4.74a       0.00c
PR15           4.45a   3.90c       1.86b
PR22           4.48a   4.38b       0.00c
PR33           4.76a   2.86d       0.00c
PR78           4.82a   3.86c       2.19a
PR84           4.41a   4.44b       1.77b
PR87           4.78a   3.70c       1.82b
Control        0.00b   0.00e       0.00c
C.V (%)        14.84   4.69        15.09

Means within column followed by a common letter are
not significantly different according to DMRT (P <
0.05).

Table 4. Yields, percentage of anthracnose incidence and disease
severity of bird chili var. Prik Kheenu in greenhouse

Treatments     No.       Fruit      Fruit    Anthracnose   Anthracnose
               fruits/   weight/    length   incidence     severity
               plant     plant      (cm)     (%)           index
                         (g)

Strep-PR22     191.7     188.65b    4.10     17.0b         3.5c
flowering

Strep-PR22     209.4     227.54ab   4.71     21.9ab        4.0bc
Seedling

Strep-PR87     207.8     186.34b    3.99     16.4b         4.0bc
flowering

Strep-PR87     237.0     212.89ab   4.23     14.7b         4.0bc
Seedling

Chemical       216.9     221.14ab   4.42     17.5b         5.0ab

Inoculated     188.7     176.69b    4.24     33.4a         5.5a
control

Uninoculated   297.3     344.85a    4.61     --            --
control

C.V (%)        16.81     26.20      6.75     29.54         9.42

Means within column followed by a common letter are not significantly
different according to DMRT (P < 0.05).

Table 5. Yields percentage of anthracnose incidence and disease
severity of long cayane var. Num Kiew in greenhouse

Treatments     No.       Fruit     Fruit    Anthracnose   Anthracnose
               fruits/   weight/   length   incidence     severity
               plant     plant     (cm)     (%)           index
                         (g)

Strep-PR22     98.0      901.37    11.73    35.9b         3.0b
flowering

Strep-PR22     106.0     953.69    12.14    24.3b         3.0b
Seedling

Strep-PR87     98.1      880.9     12.14    28.0b         3.0b
flowering

Strep-PR87     94.3      917.88    12.15    29.4b         3.0b
Seedling

Chemical       88.7      824.19    11.94    31.3b         3.5ab

Inoculated     107.9     1037.19   11.54    64.8a         4.0a
control

Uninoculated   107.2     1032.75   12.08    --            --
control

C.V (%)        20.53     18.24     6.47     26.04         8.88

Means within column followed by a common letter are not significantly
different according to DMRT (P < 0.05).
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Author:Yutthasin, Rattikan; Thummabenjapone, Petcharat; Hiransalee, Anan
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Dec 1, 2015
Words:4606
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