allelopathy is the negative effect of chemicals released by one plant species on the growth and reproduction of another .
allelopathy can affect many aspects of plant ecology , including occurrence , growth , and plant succession , the structure of plant communities , dominance , diversity , and plant productivity .
there are four main classes of chemical interactions , antibiotics ( microorganisms to microorganism ) , kolines ( plants to plants ) , marasmins ( microorganisms to plants ) , and phytoncides ( plants to microorganisms ) . in allelopathic terms , a chemical is  conveyed  to a 
allelochemicals representing numerous chemical groups have been isolated from over 30 families of terrestrial and aquatic plants .
some indian plants possessing allelochemicals are pongamia glabra ( karani ) , mathuca indica ( molarah ) , schleicheria teijuga ( kusum ) , and celastrus paniculatus ( malkanguni ) .
autotoxicity occurs when plant species releases toxic chemicals that inhibit germination and growth of same plant species .
leucaena leucocephala , the tree promoted for revegetation of soil , contains a toxic nonprotein amino acid in the leaves which inhibits growth of others but not its own seedlings .
it is commonly called gmelina and white beech ( english ) , melinaphocal ( spanish ) , gamar in bangladesh , melina gambar in india , gmelina in indonesia , yemane in philippines , and soh in thailand .
gmelina arborea is a medium - sized deciduous tree , up to 40  m in length and 140  cm in diameter , but it is usually smaller than other trees .
it can show in a wide range of diameter zones ranging from 50  m to 4500  m msl and in regions of annual rain fall ranging from 50  mm to 9500  mm .
the presence of allelopathic compounds such as polyphenols and terpenoids was analysed by tlc and gc - ms .
the extract inhibited the germination , seedling growth , and total protein content of both test crops .
the present study focused on identification of the allelochemicals by hptlc , determination of the effects of the allelochemicals on the germination of black gram , green gram , red gram , and chickpea , and analysis of allelopathic effects by conventional pot experiments .
the study also elaborated on determination of the activity of peroxidase , acid phosphatase , amylase , and catalase on various stages of germination and correlating the loss of activity to the detrimental effects of the allelochemicals present in the leaf leachates of gmelina arborea .
fresh leaves and leaf litters of gmelina arborea were collected from a six - year - old tree and stored in polyethene bags in a moisture - free atmosphere .
the seeds and plant were identified from the institute of forest genetics and tree breeding , coimbatore , tamil nadu , india .
a quantity of 50  g of fresh leaf litter was soaked in 159  ml distilled water for 24 hours at 25c .
harrington suggested that agricultural seeds retain their longevity in terms of germination efficiency if stored between 0 and 50c ; bringing down the temperature by every 5 degrees , the life of the seed doubles . in the present study ,
vigna mungo ( green gram co 7 ) , vigna radiata ( black gram co 5 ) , cajanas cajan ( red gram co 6 ) , and cicer arietinium ( chickpea co 4 ) required for the seed bioassay were procured from department of seed science and technology , tamil nadu agriculture university , coimbatore , tamil nadu , india .
ferric chloride test : 2  ml of water was added to 1  ml of each extract .
two to three drops of 10% ferric chloride solution were added and development of green color was to be observed .
liebermann 's test : 1  ml of 20% sulphuric acid was added to 1  ml of each extract followed by addition of few drops of 1% sodium nitrate solution and the tubes were observed for the formation of red color , which on dilution and in alkaline condition with sodium hydroxide turn blue .
the aqueous extract of leaf sample was centrifuged and the supernatant was collected , which was used as test solution for hptlc analysis .
a volume of 2  l of the test solution and 3  l of standard solution was loaded as 6  mm band length in the 3  10 silica gel 60f254tlc plate using hamilton syringe and camag linomat 5 instrument .
the samples ' loaded plate was placed in tlc twin trough developing chamber ( after being saturated with solvent vapor ) and the plate was developed in the respective mobile phase ( toluene - acetone - formic acid ( 4.5  :  4.5  :  1 ) ) up to 90  mm .
the plate was , then , kept in photodocumentation chamber ( camag reprostar 3 ) to capture the images under white light , uv 254  nm and uv 366  nm .
the developed plate was sprayed with a spray reagent ( fast blue b reagent ) and dried at 120c in hot air oven .
the plate was photodocumented under day light and uv 366  nm using the same photodocumentation chamber . before derivatization ,
the plate was fixed in scanner stage and scanning was done at 254  nm . the peak table , peak display , and peak densitogram were noted .
a quantity of 50  g of soil sample was taken in pots of depth of 3 inches and five seeds were sown in each pot .
triplicates were maintained ; one seed was taken from each part of the replicate and ground in 3  ml of 0.2  m phosphate buffer ( ph 7.0 ) and centrifuged at 5000  rpm for 10 minutes ; the supernatant was collected and used for biochemical analysis .
similarly , four replicates containing five seeds in each pot were maintained to calculate the total chlorophyll content , percentage of germination , germination index , vigour index , root length , and shoot length .
the number of seeds germinating was counted everyday up to the sixth day and the percentage of germination was calculated by the formula ( 1)germination%=(nt)100 , where n is the number of seeds germinated and t is the total number of seeds .
the germination index was calculated according to wiese and binning   using the formula ( 2)gi = nd , where n is the number of seedlings emerging on day 
the seed vigour index was calculated by multiplying germination percentage and seedling length ( cm ) .
the root length , shoot length , and seedling length were measured using a grading scale on sixth day after germination .
the whole plant was wrapped in aluminium foil and placed inside an oven for 48  h at 45c .
the relative water content was calculated at sixth day after germination by ( 3)relative  water  content  ( % )  = fresh  weightdry  weightfresh  weight .
the total protein content present in control seeds and treated seed lots was estimated by bradford 's method ( 1976 ) .
a quantity of 0.5  g fresh weight of green tissue from both leaf and stem was weighed .
the contents were centrifuged at 5000  rpm for 10 minutes and the supernatant was collected .
the absorbance was read at both 647  nm and 664  nm against reagent blank .
total chlorophyll content was checked by einhelling method , using the formulae ( 4)chlorophyll  a  = 13.19(abs  at  664  nm )  2.57(abs  at  647  nm )  g / g  dry  weight , chlorophyll  b  = 22.10(abs  at  647  nm )  5.26(abs  at  664  nm )  g / g  dry  weight , total  chlorophyll  = 7.93(abs  at  664  nm )  + 19.53(abs  at  647  nm )  g / g  dry  weight .
a volume of 1.9  ml distilled water , 1  ml of 0.059  m hydrogen peroxide , and 0.1  ml of diluted seed extract was added .
the decrease in absorbance was recorded at 240  nm for 2 - 3 minutes and calculated at 240  nm / min from the initial ( 45 seconds ) linear portion of the curve .
the enzyme activity was further calculated using the following relationship : ( 5)units / mg  = 240/min100043.6mg  enzyme / ml  reaction  mixture .
the reaction mixture consisted of 0.5  ml of substrate solution ( 50  mg of p - nitrophenyl phosphate was dissolved in 10  ml distilled water ) .
the ph was adjusted to 4.8 with 0.5  n naoh and 0.1  ml of suitably diluted seed extract .
a volume of 0.2 to 1  ml ( 4 to 20  mm ) of the standard ( 69.75  mg of p - nitrophenol dissolved in 5  ml distilled water to give a final concentration of 100  mm ) was diluted to 3  ml with 0.1  n naoh .
specific activity was expressed as moles of p - nitrophenol were released per minute per mg of protein .
a volume of 1  ml of 1% starch solution was taken and 1  ml of properly diluted seed extract was added to it .
the reaction was stopped by the addition of 2  ml of dinitrosalicylic acid reagent ( 1  g of dinitrosalicylic acid , 200  mg of crystalline phenol , and 50  mg sodium sulphite in 100  ml 1% naoh ) .
a volume of 1  ml of 40% potassium sodium tartrate was added and cooled under running tap water .
one unit of amylase was expressed as mg of maltose released during 5-minute incubation with 1% starch solution at 27c .
the assay mixture containing 2.5  ml of phosphate buffer , 0.2  ml of suitably diluted seed extract , and 0.1  ml of o - dianisidine ( 50  mg of o - dianisidine was dissolved in 50  ml of methanol ) was incubated at 28c in a water bath for 2 minutes .
the reaction commenced by adding 0.2  ml of h2o2 ( 0.6% ) and left for 5 minutes .
the leaf leachate of gmelina arborea was subjected to phytochemical analysis that showed a strong positive result for the presence of phenols followed by moderate indication for alkaloids .
the aqueous leaf leachate was also subjected to hptlc analysis and results are shown in figure 1 .
orange brown colored zone at day light was observed after derivatization which confirmed the presence of phenolics having an rf value of 0.51 .
quercetin had an rf value of 0.58 . hence taking into account the closeness of rf values ,
further , the ftir spectrum ascertained the presence of phenolic compounds that revealed several peaks ranging between 47.50  cm and 2305.01  cm .
this wide range of peaks could be attributed to the presence of various phytochemical constituents in the aqueous leachate representing several bond stretches .
phenols showed the characteristic c = c stretches and c  h vibration for aromatic residues .
the strong oh stress band might swamp the weaker c  h stretches band just above 3000  cm .
our earlier studies had shown similar results where various phenolic compounds like 3,4,5-hydroxybenzoic acid , 3-(4-hydroxyphenyl ) prop-2-enoic acid , and 4-hydroxy-3-methoxybenzoic acid were found to be present in the aqueous leaf leachate . similarly , several benzoic acid derivatives had been shown to possess potential allelopathic effect on red gram .
the crude aqueous leaf leachates were tested for their allelopathic effects on black gram , red gram , chickpea , and green gram .
the allelopathic effects were more predominantly seen in chickpea with 80% inhibition of germination ( table 1 ) . in the other systems ,
namely , black gram , red gram , and green gram , the percentage of inhibition ranged between 26% and 75% .
the results were also consistent with earlier report where allelochemicals from eucalyptus showed around 75% inhibition in red gram .
though only a few seeds in the chickpea seed lot germinated , the sustenance of the plantlet was not very stable as the seedling was watered with the leachate even after germination .
hence , it could be inferred that the allelochemicals of gmelina arborea were not only of inhibitory type to germination but also with retarded plantlet growth after germination .
terzi , 2008 ,   had also observed such kind of high negative inhibition where juglone on allelochemicals from walnut leaf juice significantly retarded the seedling growth of musk melon and cucumber . in the present study ,
the vigour index observed in various seed lots ranged between 33.2 and 1522 with a mean of 88.3 for black gram , between 124.3 and 1236 with a mean of 687 for red gram , and between 533.8 and 992 with a mean of 785 for green gram seed lots .
since the percentage of inhibition was too high for chickpea seed lot , decipherable vigour index was not observed ( table 1 ) .
the results indicated that the leachate influenced more vigour loss in chickpea followed by red gram , black gram , and green gram , respectively .
similar profuse vigour loss was also reported by pawar and chavan   where the allelopathic effects of eucalyptus , melia , moringa , and parthenium were observed on wheat , rice , millet , and sorghum .
the effect of allelochemicals of the leaf leachate obtained from gmelina arborea also drastically reduced the relative water content in red gram followed by the other seed lots .
this parameter was not accessible for chickpea seed lot where the percentage of survival was almost nil .
the total chlorophyll content also reduced due to allelopathic effect in treated seeds of all the seed lots ranging between 0.7 and 7.5  g / g dry weight .
similarly , the total protein content also varied between 1.02 and 0.45 in the control and between 0.25 and 0.44 in the allelochemicals treated seed lots ( figure 2 ) .
enzymes are one of the chief molecules , which are stimulated when a seed germinates ; hence it would be very appropriate in inhibiting the enzymes like acid phosphatase , catalase , peroxidase , and amylase whose expression is thought to be upregulated during seed germination . in the present study ,
activity of enzyme was found to increase as days of germination increased ; for instance , the level of peroxidase in control red gram was 0.76 units ( figure 3 ) .
this trend increased exponentially after the second day of germination reaching a peak of 1.97 units in control , after 6th day of germination .
the inhibition of phosphatase directly affects the energy metabolism and atp production and contributes to the seed vigour and retardation is visible in germination .
senna et al . , 2005 ,   had reported the favorable role of acid phosphatase in maize seeds germination where the activity of the acid phosphatase increased in 24  h of onset of germination .
similarly , the levels of prolyl aminopeptidase and acid phosphatase were higher in germinating seeds of l. esculenta .
these enzymes have the potential role in liberation of proline and other amino acids that are released by the enzymatic hydrolysis of several seed storage proteins .
matveyeva et al . , 2003 ,   had reported on several compounds like furosemide , bumetanide , and so forth , which are inhibitors of calcium cotransporters to suppress the germination rate to less than 50% .
there is potential role of emp enzymes particularly dehydrogenases in seed germination upon treatment with inhibitors .
further , the activity of these enzymes was also not stable after the plant has started the photosynthesis .
similar trends were also observed with the activity of amylase being seen only in green gram ( figure 4 ) .
this explains that in every individual type of seed different enzymes either singly or in combination are responsible for an efficient germination and plantlet establishment , all with a common role to play , namely , release of amino acids and other small molecules from the stored proteins and carbohydrates in the seed , which could be used for germination .
the present study showed that the allelopathic compounds present in the leaf leachate of gmelina arborea such as phenolics and 4-hydroxy-3-benzoic acid inhibited crucial enzymes responsible for seed germination .
compared to the studied four seed lots , the germination was inhibited in chickpea predominantly .
the relative water content and total chlorophyll content were varied from 81% to 50% and from 97% to 64% in control and treated seed lots .
the present study indicated that vigour index also varied and it was much skewed in black gram seed lots .
the present study also revealed that the allelochemicals released from gmelina arborea affected the germination and growth of black gram , green gram , red gram , and chickpea .
thus , the extract reduced the growth of economically important seeds . in the future study , gmelina arborea allelochemicals
the present study also threw limelight on the effective use of this agroforestry tree , wherein planting this tree amidst pulse related herb plantations could affect the growth of the economically viable plants .
potential plantations of gmelina arborea could be tried in uncultivable dry lands as this species can very well adapt to diversified soil and climatic conditions .
 the present work focused on identification of allelochemicals from the leaf leachates of gmelina arborea and analyzing its influence on the germination of red gram , green gram , black gram , and chickpea in terms of the levels of some important germination enzymes like acid phosphatase , catalase , peroxidase , and amylase . 
 the study showed that allelopathic effects were more predominant in chickpea with 80% followed by red gram , green gram , and black gram where the inhibition ranged between 26% and 75% . 
 the vigor index in the seed lots is also considerably reduced . 
 total chlorophyll content was also reduced by allelopathic effect in all treated seeds ranging between 0.7 and 7.5  g / g dry weight . 
 the effect of allelochemicals drastically reduced the relative water content in red gram followed by the other seed lots . the total protein content varied considerably in the control and the treated seed lots . 
 allelochemicals inhibited the expression and activity of the enzymes required for efficient germination . 
 the present study also threw limelight on the effective use of this tree , wherein planting this tree amidst pulse related herb plantations could affect the growth of the economically viable plants , but this tree can very well adapt to diversified soil conditions and rainfall zones . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions