DEVELOPMENT OF RHIZOBIUM TECHNOLOGY APPLIED FOR HYBRID ACACIA (A. auliculiformis + A. mangium) IN NURSERY AND PLANTATION Le Quoc Huy1, Nguyen Minh Chau ABSTRACT: Up to the year 2010, the National Five Million Hectar Afforestation Program in Vietnam will plant million hectars of Hybrid Acacia for both economical and ecological purposes. The research of rhizobium technology development was set to support the purposes and implementation of the project regarding the acacia species. A useful rhizobium technology has been successfully studied and developed for hybrid acacia including isolating & screening techniques in in-vitro, glass-house and nursery, multiplication & inoculum production techniques, maintenace and inoculum application techniques. Six strains of most beneficial rhizobia for hybrid acacia has been selected and developed inoculant for application in nursery and plantation trials. The growth response of hybrid acacia in nursery to the rhizobium application was as high as 200% apx. as compared to 100% in control (height, diameter, dried weight) and similarly 30 % after one year & 20%(height, diameter) after two year of planting in plantation trials. Key words: Hybrid acacia, rhizobium, strain, innoculant application

IInnttrroodduuccttiioonn Annually, there is about 120- 160 million tons of atmospheric N2 fixed in to nitrogen fertilizer forms through naturally biological nitrogen fixing process globally (Gibson, 1995). This amount of fertilizer is estimated two times as much as that of chemical prodution process in the world. In the nodule relationship between rhizobium bacteria and legume trees, the nitrogen fixing is done by the rhizobia, by which trees are supplied with nitrogen sources for their growth vice versa bacteria will obtain hydratecarbon for their esistence. This relationship is essential for nitrogen cycle in nature and for sustain yield & productivity of crop as well as for ecosystem sustainability (Macdicken, 1994. Balasundaran, 1995). The field research of rhizobium technology and its application have presently caught much attention of scientists for many different crops. Hybrid acacia, the tree crop is currently important for National Five Million hectar Afforestation Program as well as for many other afforestation projects in Vietnam. A useful rhizobium technology developed for the species could assure significant improvement of the quality & effectiveness of nursery planting stock production and plantation productivity. MMaatteerriiaallss aanndd mmeetthhooddss IIssoollaattiinngg aanndd ssccrreeeenniinngg tteecchhnniiqquueess ffoorr hhiigghh bbeenneeffiicciiaall ssttrraaiinnss ooff rrhhiizzoobbiiaa,, Rhizobium strains were isolated from nodule sources of hybrid acacia and A. mangium growing in different sites and ecological zones. The media of YMA (Yeast Manitol Agar), YMA- Red Congo &

1

Forest Science Institute of Vietnam

1

YMA- Bromthymol Blue were used for the isolation (Somasegaran vµ Hoben, 1985), by which the isolates were classified into two groups; Rhizobium, the fast growing group was with yellow acidic reaction on YMA-Bromthymol Blue and Brandyrhizobum, the slow growing group with blue alkaline reaction on the medium. Then they were selected through series of screening steps, firstly the selection of authentic rhizobia for high ability to form nodule association with the host plants growing in SNS (Seedling Nutrition Solution) (Gibson 1963, 1995, Vincent 1970, Somasegaran & Hoben 1985) in-vitro condition with “Paper Bridge” technique. The observations were the quantity and quality of nodules formed. The next step was the selection for efficacy of nodule association of tested rhizobium with host plants growing in SNS (Nitrogen-free nutrient solution) (Vincent 1970, Somasegaran & Hoben 1985) in glasshouse through method of Leonard Jar. Hybrid acacia in the experiment were inoculated with tested rhizobium, grown in Leonard Jar assembly with SNS for 8-10 weeks, by which only the plant, who formed adequately nodule association with tested rhizobium could grow well in that condition, otherwise they would be of poor growth, eventually died. Observations were numbers & weight of nodules formed, overal height (Hn), colar diameter (Do) and total dried weight (P). Rhizobium strains with high nodule association efficacy in Leonard experiment were further tested in nursery experiment. This was last step of the screening; observations were same as those of last step in Leonard. Figure 1: Overal Diagram of Rhizobium Technology and Practice for Hybrid Acacia Isolating techniques on media of Yeast Manitol Agar (YMA), YMA-Congo Red, YMABromthymol Blue

37

Screenging technique for authentic rhizobia in in-vitro, SNS medium, Paper- Bridge method for five weeks

15

Screening technique for nodule association efficacy in glasshouse: Leonard Jar Screening technique for nodule association efficacy in nursery with field soils

6

3

Maitenance techniques in slant agar and cold distilled water( 4o C) Multiplication & innoculum production of rhizobium (YMA and processed peat carrier)

Application to nursery hybrid acacia:10gr/pla.

production

Mixture: 3 strains

for

Mixture

2

Innoculum application for nurseries and plantation trials in BaV×, Hoµ Binh vµ §«ng Hµ

Invitro plants of hybrid acacia were used for materials (host plant) of experiments in In-vitro and Leonard tests in glasshouse. In experiments of nursery and plantation trials, cuttings of hybrid acacia were used as the materials. MMeetthhooddss ooff mmuullttiipplliiccaattiioonn aanndd iinnnnooccuulluumm pprroodduuccttiioonn ooff rrhhiizzoobbiiuumm,, Only the strains of rhizobium with highest nodule association efficacy were used for multiplication and production of multi-strain inoculum for application (Sharma, Sankaran, Balasundran & Sankar, 1996). The multiplication was carried out on simply modified fermenter asembly with pressureair supply going through two filters (dry & wet filters) before entering into the bottom of “fermenter”, 20 liter glass Jar was well tighly asembled with the system as the fermenter. It took about 72 hours to obtain fermented content with 109 tb/ml at temp. of 28-30oC. With conditions, after 3 days there was about 15-16 liters of the aqueous content obtained, that was enough to produce 60-80kg of innoculum. Sieved, neurturalized and sterilled fine peat was used as carrier for innoculating with aqueous rhizobium at rate of 250ml/kg to produce innoculant. It could be used after that 7 days. EExxppeerriimmeenntt llaayyoouutt,, Experiment in glasshouse was laid out in Randomized Completely Block Design (RCBD) as follows, 1 - 15 were the treatments of 15 tested rhizobium strains coded repectively, C was the control without innoculation. The lay-out of Leonard Jar experiment in glasshouse. 1 4 5 14 11 9 12 6 13 C 15 10 Replication

3 2 8 7 1

1 3 4 14 9 11 8 13 6 10 7 C Replication

5 2 15 12 2

9 C 14 5 6 2 7 12 10 1 3 4 Replication

15 8 13 11 3

14 12 2 11 C 5 1 4 6 10 15 3 Replication

9 7 8 13 4

Experiment in nursery was laid out with 6 plots, of which 5 plots with treatments of 5 respectively tested rhizobium strains coded CaI3, IIH3.1, IH1, IL1 & IQ1, selected from previous step, one control plot without rhizobium. Plantation trials were also diesigned in RCBD with 3 blocks, each block with 3 plots: 2 formulas with innoculum application of which

3

TN1: Nusery Innoculation + Plantation application one month after planting; TN2: Plantation with nusery innoculation only and 1 Control plot without any application. DDaattaa ccoolllleeccttiioonn aanndd aannaallyyssiiss:: Experiments in glasshouse and nursery were observed and measured for data of observations. The mean values of observations were obtained from the collected data in all plots for each treatment formula respectivey. To make easy for comparion among the treatment and control, all the mean values were converted to percentage (%), in which, the mean value of control was considered as 100%. “ANOVA: single factor” in Excel 5.0 was used to statistically test the significance of the differences among the mean values and of the effects of the treatments at  = 0.05 (p= 0.95). Similarly in the field trials of plantation, the data collection was done for each observation in plot and block with help of Stratified Simple Sampling method then the mean values were obtained in same way. The “ANOVA: two factors without replication” in excel 5.0 was used to statistically test the significance of the differences among the mean values and effects of treatments at  = 0.05. RReesseeaarrcchh LLooccaattiioonnss:: 33 llooccaattiioonnss Table 1: Main features of research sites and locations: Cam Quy Thung Nai Dong Ha Slopping 10-15o 25- 30o 10-15o Herbs and shrubs, Shrubs, bushes Vegetation Scattered eucalyptus, shrubs Stony (%) 30% at depth of 10- 50% at depth of 20- 30% 30cm 30cm 0-10 10-20 30-40 0-10 10-20 Soil depth 30-40 0-10 10-20 30-40 (cm) Org. carbon 2 .03 1.6 1.2 3.50 3.2 2 .4 2 2 .12 1.80 (%) .26 N (%) 0.22 0.13 0.12 0.12 0.14 0.10 0.06 0.03 0.02 P2O5 3.80 3.70 1.08 0.43 0.40 4.12 2 .90 2 .43 6.14 (mg/100g 4.8 K2O 1.60 5.60 6.10 5.46 5.00 7.23 6.03 5.60 (mg/100g pHKCl 4.14 3.80 3.80 4.55 4.40 4.10 3.81 3.91 3.91

Rhizobium innoculum was applied for Nursery production of hybrid acacia planting stock and 2 ha of plantation trial in each location of Ba Vi- Ha Tay, Thung Nai- Hoa Binh and Dong Ha- Quang Tri. RReessuullttss aanndd ddiissccuussssiioonn RReessuullttss ooff iissoollaattiioonn 35 pure strains of rhizobia were obtained from isolating nodule sources of hybrid acacia & A. mangium growing in 4 ecological zones and sites including Luong Son- Hoa Binh, Cam

4

Quy- Ba Vi, DongMo- Ha Tay and Dai Lai- Me Linh- Vinh Phuc. Two other strains were obtained from one Lab in Thailand. Out of 37 strains totally, there were 32 strains identified belonging to the group of Rhizobium- fast growing, forming distinct colony after 3 days, yellow acidic reaction on YMA-BTB, while there were only 5 strains identified belonging to slow-growing Brandyrhizobium group, blue alkaline reaction on YMA-BTB (table 2). The results were similar to those previously reported that there was about more than 90% of strains isolated from A. mangium and A. auriculiformis belonging to fast growing group of Rhizobium (Sharma, Sankaran, Balasundaran, Sanka, 1995; Ty & Thang, 1995; Lafay & Burdon, 1998; Mahobia, Mahna, 2002). This point was contributing to unravel the recent controversy said that, almost all rhizobium strains associated with tropical legume species and acacias were belong to slow growing group of Brandyrhizobium. Table No.

2. Isolating results of rhizobium strains from nodules

Codes

Origines/nodule sources

Host sources

Color reaction with YMA- CR

Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia A. mangium A. mangium Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia A. mangium A. mangium A. mangium A. mangium A. mangium A.

No color

Yello w *

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

1

IH1

Luong Son- HB

2

IH2

Luong Son- HB

3

IH3

Luong Son- HB

4

IH4

Luong Son- HB

5

IH5

Luong Son- HB

6

IIH3.1

Luong Son- HB

7

IIH3..2

Luong Son- HB

8

IB1

Da Chong- BV

9

IB2

Da Chong- BV

10

IB3

Da Chong- BV

11

IB4

Da Chong- BV

12

IB5

Da Chong- BV

13

IB6

Da Chong- BV

14

IIB1

Da Chong- BV

15

IIB2

Da Chong- BV

16

IIB3

Da Chong- BV

17

IIB4

Da Chong- BV

18

IIB5

Da Chong- BV

19

IIM1

Dong Mo - ST

YMA - BTB

No color

Group

Blue Rhizobium

*

No color

*

Brandyrhiz o Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

5

20

IIM2

Dong Mo - ST

21

IIM3

Dong Mo - ST

22

IIM4

Dong Mo - ST

23

IIM5

Dong Mo - ST

24

IQ1

Cam Quy- BV

25

IQ2

Cam Quy- BV

26

IL1

Dai Lai- Me Linh

27

IL2

Dai Lai- Me Linh

28

IL3

Dai Lai- Me Linh

29

IL4

Dai Lai- Me Linh

30

CaI.3

Da Chong- BV

31

CaI.6

Da Chong- BV

32

CaII.4

Da Chong- BV

33

CaII.6

Da Chong- BV

34 35 36

CaIII.1 CaIII.6 TL1

Da Chong- BV Da Chong- BV Thailand

37

TL2

Thailand

mangium A. mangium A. mangium A. mangium A. mangium Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia Hybrid acacia A. mangium A. mangium A. auri. A. auri. A. mangium A. mangium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

No color

*

Brandyrhiz o Brandyrhiz o Brandyrhiz o Brandyrhiz o Rhizobium

No color

*

No color

*

No color

*

No color

*

Rhizobium

No color

*

Rhizobium

No color

*

Rhizobium

No color No color No color

* * *

Rhizobium Rhizobium Rhizobium

No color

*

Rhizobium

NNoodduullee aassssoocciiaattiioonn ffoorrmmaattiioonn ooff tteesstteedd rrhhiizzoobbiiuumm ssttrraaiinnss wwiitthh iinn--vviittrroo hhyybbrriidd aaccaacciiaa ppllaanntt.. By method of “Paper- Bridge” in in-vitro, strain isolates of rhizobium were tested for ability to form nodule association with hybrid acacia plant grown in in-vitro condition after 5 weeks. There were 7 strains assessed as the “high ability” strains, they could form nodules very early & continuously, after innoculation only 1-2 weeks, the numbers of nodule were high (10-15 /plant), with big size and pink colour indicating very high active for nitrogen fixation (Gibson 1995, Somasegaran & Hoben 1985). There were 9 other strains having slower nodule formation and considered as “rather high ability”, numbers of nodule formed were not high, 4-8/plant. There were few strains with very slow process of nodule formation, 4-5 weeks after innoculation, very small numbers of nodules formed with msall size, and no pink colour, this class had 6 strains including 2 strains from Thailand. There were many others left determined to be not capable to form nodule association with the host plant.

6

From the results, it is generalized that (i) A majority of strains were high and rather high capable to form nodule association with the host plant in the test, (ii) Some strains isolated from hybrid acacia, but not well form nodule association with the host eg. IH4, IH5, IB1, IB4, etc, and vice versa some others formed well but not isolated from the host eg. IIH3.1, IIH3.2., etc (table 3). The result was similar to that reported by ACIAR about the species diversity & specificity in the association between rhizobia and acacia; out of 22 rhizobia strains isolated from each species, few strains could form fully effect nodule association with host plant respectively (Gibson, 1995). Table 3. Result of nodule formation in in-vitro between tested rhizobium strains and hybrid acacia No.

Codes

IH1 IH2 IH3 IH4 IH5 IIH3.1 IIH3.2 IB1 IB2 IB3 IB4 IB5 IB6

Level of nodule formation +++ ++ ++ +++ +++ ++ ++ ++ -

20 21 22 23 24 25 26 27 28 29 30 31 32

14

IIB1

-

33

15

IIB2

-

34

16

IIB3

-

35

17 18 19

IIB4 IIB5 IIM1

++ -

36 37 38

IIM2 IIM3 IIM4 IIM5 IQ1 IQ2 IL1 IL2 IL3 IL4 CaI.3 CaI.6 CaII. 4 CaII. 6 CaIII .1 CaIII .6 TL1 TL2 Contr ol

No.

Codes

1 2 3 4 5 6 7 8 9 10 11 12 13

Note: +++ : no formation

: high level;

++

: rather high;

Level of nodule formation +++ + + +++ +++ +++ ++ + + ++ ++ + + -

+ : low level;

-

TTeesstt rreessuulltt ooff nnoodduullee aassssoocciiaattiioonn eeffffiiccaaccyy iinn LLeeoonnaarrdd JJaarr eexxppeerriimmeenntt iinn ggllaasssshhoouussee.. 17 strains were selected for this test. Based on the assessment of designed observations it was seen that, strains IH1, CaI3, IL3, IIH3.1, IQ1 had high nodule association efficacy with hybrid acacia. All the indices (Hn, Do, P) were several times or even, ten, hundred times as much as those of control, especially the index of total dried wieght (P). In SNS medium (free-nitrogen medium), the plant that could not form nodules with rhizobium, showed very poor growth, even died. IB2 , IB3 showed no formation of nodules.

7

Table 4. Nodule association efficacy of tested rhizobium strainshiÖu with hybrid acacia in Leonard test #

1 2 2 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7

Codes

Hn (overal height) cm (% )

Do (colar diameter)

No. of nodules

cm (%)

Total dried weight (P) gr. (%)

IH1 CaI.3 IL3 IIH3.1 IQ1 CaIII.1 IB5 IL4 IIH3.2 IH2

45.5 44.0 36.2 34.3 33.3 18.2 31.5 31.0 28.5 26.8

507 489 402 381 369 202 350 344 317 297

0.23 0.25 0.20 0.19 0.18 0.10 0.20 0.15 0.18 0.13

563 625 500 469 438 244 500 375 438 313

28 58 32 36 23 49 41 13 21 13

2.06 1.98 1.62 1.43 1.21 0.18 1.16 1.13 0.73 0.60

6868 6600 5400 4767 4033 600 3867 3767 2433 1983

CaI.6

12.2

135

0.10

238

4

0.08

267

IIB5

25.9

288

0.18

458

36

0.48

1600

CaIII.6

23.8

264

0.14

344

34

0.25

833

IH3

18.2

202

0.08

189

6

0.18

600

Control

9.00

100

0.04

100

0

0.03

100

IB2

7.40

82

0.08

200

0

0.03

100

IB3

6.64

74

0.06

150

0

0.02

60

NNoodduullee aassssoocciiaattiioonn eeffffiiccaaccyy iinn nnuurrsseerryy ssccrreeeenniinngg eexxppeerriimmeenntt.. 5 qualified strains of rhizobia from last step were selected for this experiment in nursery. Result in table 5 showed that, strains of CaI3, IIH3.1, IH1 vµ IL3 were considered the best ones regarding the observations measured and they were used for multiplication and production of muti-strain innoculum for application. Table 5. Result of nuresery experiment for nodule association efficacy assessment ( Chem- Hanoi, 2000) Codes

# 1 2 3 4 5 6

CaI.3 IIH3.1 IH1 IL3 IQ1 §C

Hn (overal height) (cm) (%) 99.4 219.0 71.4 157.4 62.8 138.2 54.5 120.1 69.8 153.8 45.4 100

Do (colar diameter) (cm) (%) 0.61 217.9 0.41 146.4 0.46 164.3 0.40 177.1 0.38 135.7 0.28 100

Nodules No. 73.8 63.4 60.8 43.0 20.0 13.0

(gr.) 0.43 0.39 0.26 0.23 0.17 0.11

P (total dried weight) (gr) (%) 12.3 344.5 9.23 321.6 8.59 299.3 6.07 214.5 4.05 141.1 2.87 100

Some strains showed very high potential in the last step of Leonard experiment, but in this nursery screening experiment, they were not the most efficacious, eg. in the cases of IL3, IQ1. This might be due to their adaptability to the adverse conditions in the nursery test, i.e. pH of soil potting content, nutrition, soil humidity, compatition to soil microbes. Therefore they were lost their important biological characteristics for nodule association and fixation.

8

MMuullttiipplliiccaattiioonn aanndd pprroodduuccttiioonn ooff rrhhiizzoobbiiuumm iinnnnooccuulluummtt.. FFiigguurree 33 :: TTeecchhnniiccaall sstteeppss iinn RRhhiizzoobbiiuumm iinnnnooccuulluumm pprroodduuccttiioonn... Multiplication I: on YMA in Multiplication II: on 250 Pure and beneficial tubes and put in incubator ml conical flask with strains of o set temperature at 28 c for YMB(Yeast Manitol Broth), rhizobium stored in shaking 250 r/minute at 3-5 days depents on fast slant agar o Check innoculant for CFU’s (Colony Form Units) to assure 109 tb/gr then for storage & use within 6

growing or slow growing

temp. 28 c for 5 days.

Innoculate the mixed solution of rhizobia(109 tb/gr ) to sieved, sterilled, neutralized fine peat at rate of 250ml/kg to produce

On simple fermenter assembly with pressureair supply, two filters, 20 liter glass jar fermenter filled with YMB

Figure 4: CFU's of rhizobium in inoculant during storage

There was 600 kg of inoculum produced for the experiments in 3000 nursery and plantation trials. 2500 Inoculant was checked periodically 2000 the numbers of rhizobia per gram 15 0 0 during storage. The result showed in 10 0 0 figure 4 said that, at the 500 inoculating point, there was 0 8 s s s s s s y y h h h h h 2,5x10 a cell/1gr, 10 days later, it t t t t t d da on on on on on 0 10 m m m m m 2 4 6 9 2 increased up to more than 1 T im e o f s t o ra ge 3x109cell/1gr. and then gradually reduced the number down to 6x108 at point of sixth month, from seventh month onward, the number reduced drastically down to 8x106 cell/gr. in month of 12. This result was same as that required for standard inoculant of rhizobium(Somasegaran & Hoben 1985). 3500

Growth rate(%, control 100% )

AApppplliiccaattiioonn ooff RRhhiizzoobbiiuumm IInnooccuulluumm ffoorr nnuurreesseerryy aanndd ppllaannttaattiioonn ttrriiaallss ooff hhyybbrriidd aaccaacciiaa In nursery in Da ChongFi g u r e 5: Gr o w t h r e spo n se o f n u r se r y Ba Vi, potted hybrid acacia h y br i d a c a c i a t o t h e a ppl i c a t i o n o f plants from cutting were 250 244 239 r h i r o bi u m i n o c u l u m Hn. inoculated at rate of Do. 10gr./plant. After 3 P Control 200 months, the observation 175 data collected showed Hn: 173 171 170 167 156 244%, P: 239%, and Do: 173% 150 150 as compared to 100% in control. Similar data 100 100 100 100 collected from hybrid acacia nusery in Hoa Binh was as follows: Hn 167%; P 50 171% vµ Do 150% & nursery in Dong Ha was Hn: 170%, P: 0 175% (figure 5). So the § ¸ Ch«ng Hoµ B×nh § «ng Hµ effects of rhizobium application to hybrid

9

acacia in nursery are clearly distinct. It increased significally all growth parameters observered (P= 0.95), The response was the same, even much higher as that, reported previously for A. mangium by Ty H. X. (1995). Table

7: Responses of plantation the application of Time Measures (%) Do

growth of hybrid acaci rhizobium inoculum 1 Time 2 Hn Do Hn

Gr o w t h r a t e (% )

In plantation trials of hybrid acacia, the 1. Ba Vi trial first measure was done - TN1: applied in nursery + 123 136 114 121 about 10-12 months after plantation planting and the second - TN 2 : applied in nursery 117 120 105 113 only measure was carried out 2. Hoa Binh trial about 22-24 months after - TN1: applied in nursery + 125 125 119 120 planting. In Ba Vi plantation plantation, the first - TN 2 : applied in nursery 110 105 108 104 only measure was in 12/2000, 3. Dong Ha trial only 7 months after - TN1: applied in nursery + 145 114 127 116 planting but it showed plantation significant differences - TN 2 : applied in nursery 116 107 113 106 only between TN1 & TN2 and 100% 100% 100% 100% 4. Control (without any among them with control. application The parameter Hn was as Do: ground diameter; Hn: overal height high as 136%, Do: 123% in ; P= 0.95 TN1 (control 100%). In the second measure, one year later, the data obtained for these respective parameters was Hn: 121% & Do: 114% (control 100%). In formula TN2, the figures collected in the first measure for respective parameters were Hn: 120% & Do: 117% and respectively in the second measure were 113% vµ 105% (control 100%), much lower as compared to those in the first measure. In general, growth observations for Hn, Do of formular TN1 were all higher than those in control about 20-40% in the first measure and about 14-25% in the second measure. Similarly in formular TN2, Hn, Do were higher than those in control about 10-20% in the first measure and about 10-13% in the second measure after that one year. It was also meant that the formular TN1 increased the growth of the plantation with the two parameters measured about 10-15 % higher as compared to that in TN2 formular. Figure 6: Responses of hybrid acacia to the rhizobium application in Generally it was (Highest seenHn that, the growth responses of hybrid different natures of experiment & Do were used) acacia to the rhizobium application were drastically reduced 700 from the625 application in glasshouse experiment to nursery 597 600 application, then slightly reduced from nusery application to field plantation application, andHn also slightly reduced from Do 500 the first year measurement to second Controlyear measurement regarding plantation application (figure 6). This could be explained 400 that, In glasshouse experiment, all the conditions were well 300 controlled for experiment implementation, it was favorable for 244 219 217 the 200growth of both host plant and rhizobium as well as their 173 100

100

100

100

136 123

100

10

0

Glasshouse

Chem nursery

Bavi nursery

Bavi plantation

nodule association development. Whereas in nursery and field plantation experiments, there were increasing conditions that were adverse for these above mentioned growht and development, therefore many strains were lost their ability of nodule association formation and nitrogen fixation. Only strains with high adaptability to these adverse conditions in the field could remain their biological characteristics of nodule formation and association efficacy. This is the basic principles that the isolation and screening were based upon. CCoonncclluussiioonnss 1. A applicable technology of rhizobium has been developed and effectively applied for hybrid acacia in nursery and plantation. It presented 6 maijor technical steps in figure 1. 2. Application of multi-strain rhizobium inoculum for hybrid acacia in nursery at rate of 10 gr./plant has increased the growth up to 180-200% averagely, regarding the observations of Hn, Do and P), (control 100%) 3. Application of multi-strain rhizobium inoculum for hybrid acacia in plantation at rate of 20gr./plant has increased averagely platation growth up to 128-130% ten months after planting and 118-120% after planting 24 months, regarding the observations of Hn & Do (control 100%) 4. Formula TN1 increased averagely the growth of hybrid acacia plantation about 10-15 % higher as compared to that in TN2 formular regarding the observations of Hn & Do.; After planting 24 months, the formula TN2 still increased growth of hybrid acacia plantation about 10-12% averagely higher than that in control regarding observations of Hn & Do. References Adholeya A, Sharma M P, Bhatia N P, Tyagi C, 1997: Mycorrhizal Biofertilizer- a tool for reclaimation of wasteland and bioremediation. Alan Gibson, Training Worshop Maual on Rhizobium techniques. 1995. Guangdong- China: International Workshop on Rhizobium Techniques. Balasundaran M., 1996: Key Issues in Mycorrhizae and Nitrogen fixing symbionts research, Kerala forest research Institute, Peechi, Trichur 680653, India. Bhatiacharyya, 1999. Statistics in applied sciences. Hoµng Xu©n Tý and et al. 1995. Development of technology for intensive plantation of styrax, eucalyptus & acacia and use of legume trees for soil reclaimation and plantation yield improvement: final report of case study, KNO3.13(1992-1995). Hoang Xuan Ty, 1996: Symbiont Screening for Nitrogen Fixing Trees on Strong Acid and Acid Sulphate soils in Vietnam. In: Use of Mycorhizae and Nitrogen Fixing Symbionts in Revegetating Degraded Sites. BangkokThailand : FORSPA, Lª §×nh Kha, 1997. Hybrid acacia. Hanoi: Agricultural Publish House Loretto U. Dela Cruz, 1996: Effect of Nitrogen fixing and Mycorrhizal

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Inoculants on Growth of tree species and soil amelioration of Mt. Pinatubo affectd Grasslands. NguyÔn H¶i TuÊt, 1997. Statistical Dada Analysis on computer NguyÔn Huy S¬n, 1995. Screening rhizobia for two species of acacia growing on Bazan soil in Gia Lai Province- Vietnam. In: Vietnam Forestry Journal. 9/1995 Sharma J.K., Sankaran K.V., Balasundaran M. and Sankar S., 1996: Use of Mycorrhizae and Nitrogen Fixing symbionts in Reforestation of Degraged acid soils in Kerala. In:Use of Mycorhizae and Nitrogen Fixing Symbionts in Revegetating Degraded Sites. Bangkok- Thailand: FORSPA, Singh Sujan, 1998: Interaction of Arbuscular Mycorrhizae Fungi with Nodule Forming Nitrogen Fixing Organisms- Part II. Factors affecting efficiency of Tripartile System. Somasegaran P. and Hoben H.J., 1994. Methods in Legume RhizobiumTechnology, Niftal Project University of Hawaii, USA. TrÇn CÈm V©n, NguyÔn Xu©n Dòng, Lý Kim B¶ng, 1995. Nodule formation and some biological characteristics of rhizobium strains associated with Acacia mangium. In: Biology Journal of Vietnam, 3/1995 Vincent, J.M. 1970. A manual for practical study of the rood nodule bacteria, Oxford, UK: Backwell Scientific Publications

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TuyÓn chän kh¶ n¨ng céng sinh b»ng Ph-¬ng ph¸p “CÇu giÊyèng nghiÖm”

TuyÓn chän hiÖu lùc céng sinh b»ng ph-¬ng ph¸p B×nh Leonard trong nhµ kÝnh

Lªn men nh©n sinh khèi vi khuÈn rhizobium b»ng “d©y truyÒn kü thuËt c¶i tiÕn ®¬n gi¶n”

KiÓm tra nghiÖm thu m« h×nh rõng trång keo lai bãn chÕ phÈm rhizobium (Ba V× 6/2002)

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