Plant and Soil 41, 279-286 (1974) Ms. 2336

S T U D I E S ON T H E N O D U L A T I O N AND

N I T R O G E N F I X A T I O N BY I N F E C T E D

L E G U M I N O U S P L A N T S

I. EFFECT OF ARHAR MOSAIC VIRUS INFECTION ON

NITROGEN VALUE, NODULATION AND NITROGEN

FIXATION BY SOME PULSE CROPS

by R. SINGH and T. P. MALL

Botany Department, Gorakhpur University, Gorakhpur, U.P. (India)

SUMMARY

The effect of arhar mosaic virus strains (ASM & AMM) infection on the growth, nitrogen value, nodulation and nitrogen-fixing capacity of Vigna s inensis Savi (cowpea), Phaseoh,s aureus Roxb. (mung) and Phaseolus mungo L. (nrd) has been studied. Both the strains lowered the growth and fresh weight of infected plants in comparison to healthy plants. The dry weight, of ASM and AMM strains-infected mung and urd plants, increased but it was reduced in diseased cowpea plants. The anhar mosaic virus infection de- creased the number, weight and size of nodule in cowpea and mung but increased the nodule number and fresh weight in urd plants.

The total nitrogen value of the ASM and AMM strains-infected cowpea and mung plants was lower than in healthy but it is increased in diseased nrd plants. The arhar mosaic virus strains reduced the nitrogen fixation capacity by infected plants in comparison to their healthy counterparts.

INTRODUCTION

Tile studies on the virus-infected legumes, specially on their nitrogen value, nodulation and nitrogen fixation is limited and only confined to soybean 16 17 and Dolichos 11. These studies mainly cover the effect of virus infection on nodulation and nitrogen fixation. Such studies on most of the important pulse crops are lacking. The present investigation was, therefore, undertaken to study the effect of arhar mosaic virus strains (ASM and AMM) on growth, nitrogen value, nodulation and nitrogen fixation by cowpea, mung, and urd plants.

280 R. SINGH AND T. P. MALL

MATERIALS AND METHODS

All the experiments were conducted in insect-proof glass house. Cowpea (Vigna sinensis Savi) Cv. Fodder Type-2, mung (Plmseolus aureus Roxb.) Cv. Sheela and urd (Phaseolus mungo L.) cv. Type 65 were taken as host plant in the present experiment. Arhar mosaic virus isolates (ASlVI and AMM) origin- ally isolated from field grown arhar (Ca]anus cajan (L.) Millsp.) and maintain- ed in Ca]anus cajan Cv. s-g were taken as virus. Both the strains only infect plants of leguminosae family. Strain ASiV[ is a severe strain having D E P 1 : I000,000, T IP 80°C and longivity 'in vitro ' t 1 days while the other (AMM) comparatively milder strain's D E P 1:1000,000, T I P 60°C and longivity 'in vitro ' 16 days at temperature (lVfin-48-62 Max 84-100°F). The crude infective sap was obtained from infected arhar leaves by macerating it in a mortar and squeezing the pulp through muslin cloth. The crude sap was diluted 1 : 10 with distilled water, which served as virus inoculum. The inoculum of both isolates was prepared separately. Before seeding the seeds were treated with a sus- pension of Rhizobium which was obtained from U.P. Agricultural Research Insitute, Kanpur (India). Seedlings were grown in earthenware pots contain- ing a mixture of sterilized sand, loam and compost (1:1:2) and were watered uniformely and regularly. Each pot was filled with 5 kg of above mixture. Five plants per pot were grown. Sixty such pots of each legume were taken and divided into three sets of 20 pots each. Seven days old seedlings of each legume of first and second set were inoculated separately with ASM and AMM strains respectively. The third set of seedlings was left as control. At the time of inoculation, leaves were dusted with 400-mesh carborundum powder. The inoculated leaves were washed immediately with tap water. The experimental plants were harvested after 45 days of the inoculation. At the time of harvest growth data was taken as described by S i n g h and B h a r g a v a la. At this t ime the nodules of each harvested plant were collected by removing the soil in running water through a 40-mesh screen and collecting the nodules from the screen. Those still adhering to the roots were picked off individually. Nodules were surface dried with filter paper, sealed in a small plastic bag and stored at 4°C. Nodules from each group of plants were counted, weighed and its volume was taken to know the size.

The total nitrogen was determined from dried plant material (80°C). The material was digested by the micro-method of D o n e e n 4 and the amount of nitrogen was estimated by a colorimetric procedure described by S n e l l and S n e l l 14 by using Hilger Pat tern Biochemical Absorptiometer using filter No. 43. The nitrogen of pot soil was estimated just before seeding and then at the time of plant 's harvest. For finding out the soil nitrogen the pot soil of each set of plant was thoroughly mixed and only 10-g of sample was taken for nitrogen estimation. This was estimated by the colorimetric procedure des- cribed by M i s r a s. The nitrogen value of plants is the total sum of the nitro- gen present in different parts of the plants and the nitrogen added for fixed to the soil is obtained from the difference between the nitrogen present in the pot soil at the time of harvest minus nitrogen of pot soil at the time of seeding.

V I R U S I N F E C T I O N A N D N I T R O G E N F I X A T I O N B Y P U L S E C R O P S . I 2 8 1

E X P E R I M E N T A L R E S U L T S

Results of Table 1 indicate that arhar mosaic virus strains (ASM and AMM) reduced the shoot height, root length and total fresh weight of infected cowpea, mung and urd plants in comparison to healthy. These strains increased the total dry weight of infected mung and urd plants but they lowered it in diseased cowpea. As the ASM was a severe strain it induced more reduction than AMM strain which was a milder one. ASM and AMM strains infection reduced the number, volume, fresh and dry weight of nodule in

T A B L E 1

Effect of arhar mosaic virus on growth of urd, mung and eowpea plants

Treatment Urd * M u n g * C o w p e a *

Height of shoot (cm)

Healthy 36,6 - - 29.7 - - 69,9 --

A S M 34.4 - - 0 6 . 0 25.0 - - 15.8 63.0 - - 9 . 9

A M M 35.6 - - 0 2 . 7 26 .7 -- 10.1 51.9 - - 2 5 . 8

Length o/root (cm)

Healthy 19.8 -- 16.5 - - 25.7 --

A S M 19.8 00.0 16.2 - - 0 1 , 8 21.5 -- 16,3

A M M 19.0 - - 0 4 . 0 15.5 - -06 .1 21.2 -- 17.8

Fresh weight o/shoot (g)

Healthy 16.45 - - 07.13 -- 38 .22 --

A S M I4 .80 -- 10.0 03.46 - - 8 1 . 5 27.08 - - 2 3 , 3

A M M 15.80 - - 0 3 . 9 06 .04 -- 15.3 22 .50 - - 3 6 . 3

Fresh weight o/root (g)

Healthy 01.00 -- 00.42 -- 01.38 - -

ASIVi 01 .44 + 4 4 . 0 00 .14 - - 6 6 . 7 01.16 -- 15.9

A M M 01 .20 + 2 0 . 0 00 .20 - - 5 2 . 4 01.33 - - 0 3 . 6

Dry weight o~ shoot (g)

Healthy 02.58 -- 01 ,32 -- 04.45 --

A S M 02,72 -605.1 01.54 -6 16.7 03.28 - - 2 6 . 3

A M M 02.60 -604 .7 01 .52 -6 15.2 01.07 - - 7 6 . 0

Dry weight o/root (g)

Healthy 0 0 . 2 0 - - 0 0 . 1 2 - - 0 0 . 3 3 - -

A S M 00.17 - - 1 5 . 0 00 .06 - - 5 0 . 0 00 .30 - -9 .1

A M M 00.20 00.0 00 .07 -- 41 .7 00 .32 3.0

* % increase (-6) o r dec rea se ( - - ) over healthy.

282 R. S I N G H A N D T. P . M A L L

urd plants. In the mung plants both strains differ in their trend of reaction. Though these strains lowered the number of nodule and its volume but ASM strain increased fresh and dry weight of the mung nodules while AMM strain reduced the mung nodule weight (Table 2).

T A B L E 2

Ef fec t of a r h a r mosa i c v i ru s on n o d u l a t i o n of urd , m u n g a n d cowpea p l an t s

T r e a t m e n t U r d * M u n g * Cowpea *

No. o] nodules plant H e a l t h y 49.7 -- 06.8 -- 19.4 --

ASM 50.1 + 08.0 01,8 -- 73,5 13.5 - -30 .4

AMM 49.8 + 0 2 . 0 04.1 - -39 .7 17.2 -- 11.3

Fresh weight nodule (g x 10 -2)

H e a l t h y 0.420 -- 0.140 -- 2.010 --

ASM 0.525 + 2 9 . 7 0.240 + 7 1 . 4 0.960 - -52 .2

AMIV[ 0.480 + 1 4 . 2 0.140 00.0 0.780 - -61 .2

Dry weight nodule (g x 10 -2) H e a l t h y 0.070 -- 0.023 -- 0.300 --

ASM 0.072 + 0 2 . 8 0.030 + 3 0 . 4 0.160 - -46 .7

AMlV[ 01072 + 0 2 . 8 0.022 - -04 .3 0.230 - - 2 3 . 3

Volume~nodule (ml x 10 -2) H e a l t h y 0.394 -- 0.300 -- 2.000 --

ASM 0.393 --00.3 0.280 --06,6 0'830 --58.5 AMM 0.393 - -00 .3 0.300 00.0 0.714 - -64 .3

* % inc rease ( + ) or dec rease ( - - ) o v e r hea l ty .

A perusal of the Table 3 indicates that arhar mosaic virus strains increased total nitrogen percentage in all urd parts, while lowered it in mung shoot with an increase in root and nodule. In ASM and AMM strains-infected cowpea shoot, root and nodule, the nitrogen percentage was higher than in healthy cowpea.

The nitrogen value in infected urd plants has increased but in infected cowpea and mung plants it is decreased in comparison to healthy one. Before seeding the pot soil contained 0.22 per cent of nitrogen but at the time of harvest percentage of soil nitrogen was raised. The maximum increase was noted in the pot soil which contained healthy cowpea plants followed by mung and urd. Virus infected plants fixed low amounts of atmospheric nitrogen in

V I R U S I N F E C T I O N A N D N I T R O G E N F I X A T I O N B Y P U L S E C R O P S . I 2 8 3

T A B L E 3

Ef fec t of a r h a r m o s a i c v i r u s on n i t r o g e n c o n t e n t * * of p l an t s (urd, n lung and cowpea) a n d

soil

T r e a t m e n t U r d * M u n g * Cowpea *

Percent total nitrogen in shoot

H e a l t h y 1.88 -- 2.42 -- 1.46 --

ASM 1.90 + 01.1 2.32 -- 04.1 1.36 -- 06.8

AMM 1.88 00.0 2.32 -- 04.1 1.72 + 15.1

Percent total nitrogen in root

H e a l t h y 0.84 -- 1.46 -- 1.18 --

ASM 1.06 + 2 6 . 2 2.36 + 6 1 . 6 2.32 + 9 6 . 0

AMM 1.05 + 25.0 2.34 + 60.3 2.10 + 78.0

Percent total nitrogen in nodule

H e a l t h y 2.32 -- 2.20 -- 2.32 --

ASM 2.76 + 19.0 2.50 + 8 . 7 2.90 + 2 5 . 0

AMM 2.58 + 11.2 2.45 + 6 . 5 2.54 + 0 9 . 4

Nitrogen value

H e a l t h y 0.0504 -- 0.0336 -- 0.0689 --

ASM 0.0535 + 6 . 1 5 0.0300 - -10 .7 0.0505 - -26 .7

AMM 0.0509 + 0 . 9 9 0.0367 - -09 .2 0.0252 - -63 .4

Percent total soil nitrogen at t ime oJ harvest

H e a l t h y 0.64 -- 0.84 -- 0.96 --

ASM 0.52 - -25 .0 0.57 - -20 .2 0.86 -- 10.4

AMM 0.60 - -06 .3 0.74 -- 11.9 0.90 - -06 .6

Total nitrogen added to the soil

H e a l t h y 0.42 -- 0.62 -- 0.74 --

ASM 0.30 - -28 .6 0.35 - -43 .5 0.64 -- 13.5

AMM 0.38 -- 09.5 0.52 -- 16.1 0.68 -- 00.1

* % inc rease ( + ) or dec rease ( - - ) o v e r hea l t hy .

** N i t rogen ]100 m g d r y wt .

comparison to healthy legumes. With diseased plants, highest amount of nitrogen was fixed by AMM strain-infected cowpea plants and the lowest by ASM strain-infected urd plants.

D I S C U S S I O N

The arhar mosaic virus infection reduced the growth of cowpea, mung and urd plants. Stunting of the growth is the most common symptom produced by virus infection 2. A variation in fresh and

284 R. SINGH AND T. P. MALL

dry weights of infected plants existed. A decreased fresh weight of infected mung and urd plants with increased dry weight was recorded while a lower fresh and dry weight ratio was noticed in diseased cowpea plants. As concerns, the effect of viruses on legumes fresh and dry weight ratio, both increases ~ and decreases a 12 have been reported. The range of growth reduction varied with the different leguminous species. It is due to differences in susceptibility and resistance which affects the growth behaviour. Such observation finds support from other workers also 1.

The virus infection increased the number, weight and volume of nodules in urd plants and reduced in cowpea and mung as compared to their healthy counterparts.

Like response of infected urd nodulation, R a j g o p a l a n and Ra j u 11 also reported that dolichos enation mosaic virus infection increased the number and size of nodules on Dolichos lablab L. plants. Tu, F o r d and Q u i n i o n e s 16 studied the effect of soybean mosaic virus infection on the nodulation of soybean cultivars and noticed that in infected cultivars, the number size and weight of nodules were reduced. Some other factors such as ineffective symbiosis are characterised by small nodules that fail to grow to normal size because the degeneration that starts in the bacterial region quickly 10. Since arhar mosaic virus infection decreased the nodule number in cowpea and mung and increased the nodule number in urd plants, the process is probably a more complicated physiological than a simple reduction of photosynthesis.

The total nitrogen value increased in the infected urd plants but decreased in cowpea and mung. Reports on nitrogen contents of the legumes infected with viruses are somewhat inconsistent. Some legumes with virus infection show an increase 7 9 in nitrogen while in others there is a decrease 6. The higher nitrogen value in infected urd plants can be explained on the observations of T a k a h a s h i and Ish i i 15 who indicated that infected plants contain in addition to virus, varying amounts of abnormal proteins serologically related to tobacco mosaic virus.

Total nitrogen content in nodules is generally believed to be most reliable for determining the effectiveness of nodules in legumes. Though nodules of urd and mung infected plants contained higher nitrogen than their healthy counterparts, but the nitrogen available to the soil was lower than healthy urd and mung grown pot soil.

VIRUS INFECTION AND NITROGEN FIXATION BY PULSE CROPS. I 285

Tu, F o r d and G r a u 17 also found higher nitrogen content in the SMV-infected soybean nodules and R a j g o p a l a n and R a j u 11 in D. lablab. They believe that the higher nitrogen in the nodules is due to insufficient utilization of nitrogen by the SMV-infected soy- bean. The nodule nitrogen in infected cowpea and urd was less in comparison to their healthy counterparts. The results of the present finding indicate that the total nitrogen in nodule is dependent on the Rhizobium population in the nodule which may be influenced by virus infection. The host which influences the virus multiplication as well as Rhizobium association plays a specific role. This seems to be a reason for higher or lower nodule nitrogen in different host with the same virus strain.

Increase in nitrogen level in the soil at the time of harvest is probably due to the fact that the rate of nitrogen fixation exceeds rate of consumption by the plant which results the accumulation of nitrogen in the soil. The arhar mosaic virus strains infection caused decrease in the nitrogen level of the soil. It also effects nitrogen fixation by the nodular bacteria and thus lowers down the nitrogen level of soil as compared to healthy one (Table 3).

ACKNOWLEDGEMENTS

The au thors are t hankfu l to Prof . K. S. B h a r g a v a , H e a d D e p a r t m e n t of Bo tany , The Univers i ty , Gorakhpur (U.P.) Ind ia for l abora to ry facili t ies and to S h r i S. N. G u p t a for going t h r o u g h the manuscr ip t . The junior au tho r is t hankfu l to the Un ive r s i t y for f inancial assis tance.

Received September 20, 1973

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