calcium deficiency dark-grown seedlings phaseolus lof ph. control plants were grown in cotton wool...

Plant Physiol. (1971) 47, 799-804

Calcium Deficiency of Dark-grown Seedlingsof Phaseolus vulgaris L.1

Received for publication October 23, 1970

KATiE HELMSDivision of Plant Industry, Commonwealth Scientific and Irdustrial Research Organization, Canberra, Australia

ABSTRACT

Hypocotyl collapse in dark-grown seedlings of Phaseolusvulgaris cv. Pinto was due to calcium deficiency. There was noevidence of an associated pathogen. The number of seedlingswith hypocotyl collapse decreased and the mean hypocotyllength increased when increasing levels of calcium (0-100micrograms per gram) were supplied in an external nutrientsolution to seedlings grown under sterile conditions.When seedlings were supplied with a complete nutrient solu-

tion, containing calcium at 100 micrograms per gram, butminus potassium, magnesium, sulfur, nitrogen, or phosphorus,occasional plants developed hypocotyl collapse symptoms;however, the lengths of hypocotyls varied little from those ofcontrols grown in complete nutrient. When the calcium levelin the deficient nutrient solutions was raised to 200 micro-grams per gram, the number of plants with hypocotyl collapsewas reduced markedly.With complete nutrient solution minus calcium, seedlings

developed symptoms of calcium deficiency irrespective of seedsize, i.e., irrespective of whether or not the seed contained atotal calcium content that was low or relatively high.An increase in hypocotyl length in response to an external

supply of calcium was obtained with five cultivars of Phaseolusvulgaris L. and with one of Soja max Piper. A similar responseto calcium was obtained for epicotyl growth of a cultivar ofVicia faba L., but not for a cultivar of Pisum sativum L.

When Phaseolus vulgaris L. cv. Pinto was grown in the darkat a constant temperature of 25 C in vermiculite moistenedwith distilled water, some seedlings developed necrosis of thehypocotyl and failed to emerge, whereas others developed agrey translucency of the hypocotyl soon after emergence andsubsequently collapsed. The nature of the symptoms suggestedthat they could be due to damping off, a widespread pre- andpostemergence seedling disease which is incited by severalfungal organisms (8, 16). It also was possible that the diseasewas due to a bacterial pathogen. However, data in this paperwill show that the symptoms were caused by calcium defi-ciency.

Several reports show that incidence of seedlings with symp-toms resembling damping off can be reduced by treatment ofsoil or seed with calcium. Albrecht and Jenny (1) reported thefrequent occurrence of damping off of seedlings of soybean

I Part of this work was done while the author held an Alexandervon Humboldt Fellowship.

despite sterilization of media and surface sterization of seeds.They postulated that a physicochemical condition of the soilmight be responsible. Their data showed that calcium was animportant factor in control of the disease, but did not excludethe possibility that a pathogen was implicated. Later, Angell(2) reported that liming of soil reduced the occurrence of aphysiogenic disease of poppy seedlings which resembled seed-ling blight-a general term used to describe symptoms of plantdisease which may include sudden wilting (8). More recently,Clark and Kline (6), Williams (17), and Williams et al. (18)described and illustrated a disease of bean seedlings which ap-peared in seed germination tests and also in field tests. Forsome varieties of bean the incidence of the disease was reducedby treatment with calcium. The possibility that a pathogen wasimplicated in disease development was not excluded and thecause of the disorder was not identified. A comparison of thedata of Williams et al. with those in the present paper suggestthat it was due to calcium deficiency.

Dark-grown bean seedlings are used in many physiologicaland biochemical experiments, and it was unexpected that theyshould be affected by a nutritional deficiency. Generally, it isassumed that the cotyledons provide adequate nutrients tosupport growth for a number of days. Burstrbm (4) stated, forexample, that hypocotyls employed in routine growth tests areamply supplied with calcium from their seed.

Although wilting of petioles, peduncles, and stems is associ-ated with calcium deficiency in some plants (9), the author isunaware of a previous report which shows that wilting of hy-pocotyls can result from calcium deficiency.

This paper describes experiments designed to identify thecause of the hypocotyl collapse symptom in Pinto bean seed-lings and to learn if it occurs in other legumes grown undercomparable conditions.

MATERIALS AND METHODS

Except when otherwise stated, seed of Phaseolus vulgaris L.cv. Pinto was used. This was obtained from the Haney SeedCompany, Twin Falls, Idaho.Growth of Seedlings under Nonsterile Conditions. Seedlings

were grown singly in 7-cm pots using vermiculite, perlite, orcotton wool as support. The pots were watered with de-mineralized water or with nutrient solution. Before sowing, theseeds were sterilized for 5 min in 0.1% HgCl, and then rinsedin three or four changes of demineralized water. The pots werekept in the dark at 25 C. Seven days after sowing, the seed-lings were rated for disease incidence and the lengths of thehypocotyls were measured. Except when otherwise stated, 10seeds were sown for each treatment in each experiment.Growth of Seedlings under Sterile Conditions. Each seedling

was grown separately in a glass tube of dimensions 1 X 6inches, fitted with a cotton wool stopper. At the base of each

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Plant Physiol. Vol. 47, 1971

tube was a piece of cotton wool weighing approximately 0.45 g.This was moistened with 5 ml of distilled water or nutrientsolution. Above this was a disc of filter paper of 1-inchdiameter. Determinations of calcium showed that, under theconditions of the experiment, the autoclaved cotton wool (to-gether with the filter paper disc) could release 0.055 tug/gcalcium into 5 ml of distilled water. The stated levels of calciumin the experiments to be described are exclusive of the baselevel of calcium in the tubes due to the presence of cotton wool.The seed was sterilized for 5 min in 0.1% HgCl,, rinsed inthree or four lots of sterile distilled water, and then placed inthe autoclaved tubes between the cotton wool and the paperdiscs. The tubes were kept in the dark at 25 C for 7 days, whenthe seedlings were examined for disease incidence and thelengths of the hypocotyls were measured. In all experimentsthere were 12 replicates for each treatment.

FIG. 1. Pinto bean seedlings grown under nonsterile conditionsin darkness at 25 C in "poor" vermiculite watered with distilledwater. Left: Normal seedling; right: three seedlings with hypocotylcollapse.

Nutrient Solutions. Hoagland's type nutrient solutions wereused. The basic solution (complete nutrient) contained Ca(NO)2,5 mM; KNO°, 5 mM; KH2PO,, 1 mM; MgSO,, lmM. For all ex-periments made under sterile conditions and for those requiringcomplete nutrient solutions minus one major element, the com-plete nutrient solution was used at half-strength. For the solu-tions deficient in a particular element, calcium was replaced bythe same number of milliequivalents of potassium; potassiumand magnesium were replaced by sodium; nitrate, phosphate,and sulfate were replaced by chloride.

Estimation of Calcium. Determinations of levels of calciumin experimental material were kindly made by Dr. D. J. Davidof the Division of Plant Industry, CSIRO, using atomicabsorption spectrophotometry (7).

RESULTS

Tests to Learn If the Disease Was Due to a Pathogen. Eightsamples of Pinto bean seed were tested from western UnitedStates and two from Australia. All produced seedlings withhypocotyl collapse symptoms when grown under nonsterileconditions in some lots of vermiculite watered with distilled ordemineralized water (Fig. 1).The occurrence of a high incidence of the disease in some

lots of vermiculite, but not in others (0-100%), suggested thata pathogen might be present in vermiculite from some sources.Experimental evidence was not in agreement with this possi-bility. Disease development in "poor" vermiculite occurredwhether or not it was autoclaved or fumigated with brominegas and whether or not the seed was treated with a seed pro-tectant, surface sterilized with HgCl,, or fumigated withbromine gas. The results suggested that if a pathogen was re-sponsible for the disease, it was not inactivated by sterilizationof the vermiculite or by standard methods of surface sterili-zation or fumigation of the seed. However, it was possible thatthe disease was due to a pathogen borne within the seed andtherefore unaffected by surface sterilization. Such pathogenscan be affected markedly by the physiological status of the host,which in turn can be influenced by nutrition (12).

Microscopic examination of diseased hypocotyls gave noevidence of fungal infection. No or few bacteria were detectedby phase contrast microscopy in exudate from vascular bundlesof diseased hypocotyls. However, bacteria were readily seen inassociation with collapsed tissue. Isolates of these bacteria wereidentified as Pseudomonas testosteroni (Marcus and Talalay).This bacterium was isolated previously from soil (14). In-fectivity tests provided no evidence that it was the causal agentof the present disease.

Attempts were made to isolate an organism from the hy-pocotyls of 7-day-old seedlings grown in tubes under sterileconditions. Tissue pieces from diseased and adjacent areaswere teased into tris buffer, pH 7.2. After 2 hr, droplets of thisbuffer as well as tissue pieces were placed on nutrient agar oron a modified White's medium in agar. Incubation was at 30 C.The plates remained sterile.From these experiments no evidence was obtained that the

disease was due to a fungus or a bacterium.Nutrition in Relation to Disease Development. Tests with

leachates from a vermiculite in which plants grew well reducedthe detectable disease incidence among seedlings grown in"poor" vermiculite. A possible explanation was that some lotsof vermiculite lacked certain nutrients essential for vigorousseedling growth. If this were correct, one could expect that thedisease would develop in seedlings grown on substrates withlittle or no available nutrients. In the following test the inci-dence of hypocotyl collapse was compared in seedlings grownon cotton wool, in perlite, and in "good" vermiculite wateredwith demineralized water or with complete nutrient. Table I

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CALCIUM DEFICIENCY OF PHASEOLUS SEEDLINGS

Table I. Intcidence of Hypocotyl Collapse Symptomsin Bean Seedlings'

The seedlings were grown under nonsterile conditions at 25 Con cotton wool, in perlite, and in "good" vermiculite wateredwith complete nutrient or with demineralized water.

Cotton Wool Perlite Vermiculite

Nu- Demin- Nu Demin- Nu- Dmn

trient eralized trient eralized trient eralizedrin water water water

Mean %0 diseased seed- 0 87.0 0 50.0 0 0lings

Mean length of hypo- 13.1 4.1 13.4 3.3 10.7 10.7cotyls (cm)

1 Mean data for 30 plants per treatment (three experiments).

lapse did not occur in seedlings grown in any of the threegrowth media watered with nutrient solution, nor in "good"vermiculite watered with demineralized water. Apparentlythis vermiculite in itself contained sufficient nutrients forvigorous seedling growth.

Since the pH of the media watered with demineralized waterwas higher than that of media watered with nutrient solution,it was possible that high pH contributed to disease develop-ment. This was tested as follows: Seeds sterilized in HgCl, weregerminated in the dark at 25 C in open containers containingcotton wool moistened with 0.06 M Sorenson's buffer at a rangeof pH. Control plants were grown in cotton wool moistenedwith demineralized water or with complete nutrient. In atypical experiment in which 10 seeds were grown on cottonwool moistened with buffer of pH 5.2, 6.2, 7.2, and 8.0;demineralized water of pH 5.3; and complete nutrient of pH5.0, numbers of plants with collapsed hypocotyls were 8, 9, 9,8, 9, and 0, respectively. There was no evidence that pHaffected the hypocotyl collapse symptom.

These data, together with the failure of attempts to isolatea pathogen from seedlings grown under sterile conditions,suggested that the disease could be due to a nutritional defi-ciency.

Calcium Deficiency in Relation to Disease Development.Since wilting of petioles, peduncles, and stems sometimes isassociated with calcium deficiency symptoms (9), it was con-sidered that the bean seedling disease could be due to a defi-ciency of calcium. A visual comparison of arc emission spectro-grams showed that the level of calcium in a vermiculite whichsupported normal seedling growth was about 0.2%, whereasthat of two vermiculites in which all seedlings developed hy-pocotyl collapse symptoms was about 0.03 and 0.09%.

Seedlings were grown under sterile conditions either in half-strength nutrient or half-strength nutrient minus calcium. Ineach of several tests, all seedlings grown in the calcium-defi-cient nutrient developed hypocotyl collapse symptoms, whereasnone developed these symptoms in the half-strength nutrient.Symptoms of plants grown in the calcium-deficient solution(Fig. 2) closely resembled those of plants grown in "poor"vermiculite (Fig. 1). Hypocotyls of seedlings developed a greytranslucency and soon collapsed. The translucency often wasconfined to the hook region which sometimes developed

cJ

00

0

en

z

FIG. 2. Pinto bean seedlings grown under sterile conditions indarkness at 25 C. Left: Half-strength nutrient solution; right: half-strength nutrient solution minus calcium.

shows that collapsed hypocotyls developed from seed sown oncotton wool and on perlite, only when these were watered withdemineralized water, and that disease development was as-sociated with reduced growth of hypocotyls. Hypocotyl col-

i

z

CL

C)

L)

0LL'S

4L 60

LEVEL OF CALCIUM ( pg/g)

FIG. 3. Effect of calcium level on incidence of hypocotyl col-lapse symptoms and on length of hypocotyls. Pinto bean seedlingswere grown under sterile conditions in darkness at 25 C in half-strength nutrient solution with several levels of calcium. Mean datafor three experiments.

801Plant Physiol. Vol. 47, 1971




exudate on its surface. Sometimes an enlarged collar regiondeveloped on the hypocotyl between the cotyledons and thecollapsed region; occasionally, brown flecks developed on thehypocotyl. Transverse sections made in the collapsed regionshowed that the walls of the xylem vessels were normal inform but brown in color and that both the cortex and the pithcells had collapsed. Roots were stunted and usually brown.

Disease development under sterile conditions was examinedin relation to level of calcium supplied. Disease incidence de-creased and hypocotyl length increased with increase in con-centration of calcium in the external solution (0-100 .tg/g)(Fig. 3). The withholding of calcium from the nutrient solutioncaused almost 100% of seedlings to develop hypocotyl col-lapse symptoms. It reduced the mean length of hypocotyls from17.7 to 3.3 cm. One can conclude that an external supply ofcalcium was required for normal seedling growth.

Hypocotyl Collapse in Relation to Seed Weight and CalciumContent of Seed. Seed was divided by weight into four lotsA, B, C, and D as shown in Table II. It was sown in steriletubes containing half-strength nutrient minus calcium. Al-though the total amount of calcium per seed in lots A to Cincreased with increase in seed weight, there was no evidenceof an associated decrease in incidence of hypocotyl collapse

Table II. Hypocotyl Growth in Relationi to Weight anid CalciumConltenit of the Seed'

-No. ofRangefSeeCalcim~ Toal Ca Seedlings 'MeanWaneight rCalCiUMTotalCSaee w ith iLength ofNN'eigh t per Seed Hypocotyl Hypocotyls

Collapse3

g C, g X 10-4iO'A 0.100-0.200 0.067 1.14 21 4.1B 0.225-0.325 0.113 2.30 23 3.7C 0.350-0.450 0.132 5.43 22 3.8D 0.475-0.575 0.097 4.78 23 3.7

Seedlings were grown under sterile conditions in half-strengthnutrient minus calcium.

2 Mean level of calcium per seed (C- dry weight measured forsix seeds of each seed lot).

I Twenty four seedlings were examined for each treatment (twoexperiments). Three, one, and two seedlings in lots A, B, and C,respectively, were abnormal, but did not show symptoms ofhypocotyl collapse; in lot D, one plant was normal.

Table 1I. Hypocotyl Growth of Pinito Bealn Seedlintgs in NlutrienitSolIttioni from?1 wvhich Potassilum?, Magnlesilum, Su(lflur, Nitrogenl,

or Phosphorus was WithheldPlants were grown under sterile conditions in half-strength

nutrient solution with either 100 or 200,g 'g calcium. Data for 24plants per treatment (two experiments).

Com-plete -K_ _\g -S -N -P

trient

100 pg g CalciumTotal no. of plants with hypo- 1 1 3 0 5 1

cotyl collapse symptomsMean hypocotyl length (cm) 17.5 16.71 16.2 18.4 14.6 19.7

200 pgjg CalciumiiiTotal no. of plants with hypo- 0 0 0 0 1 0

cotyl collapse symptomsMean hypocotyl length (cm) 17.5 17.8 18.1 18.016.617.3

symptoms or an associated increase in mean hypocotyl length.Similar results were reported by Williams et al. (18) in respectto hypocotyl collar rot of bean seedlings.

Potassium, Magnesium, Sulfur, and Nitrogen Deficiencies inRelation to Symptoms. Tests were made to learn if hypocotylcollapse and reduction in hypocotyl length were specific forcalcium deficiency or whether they could be produced by with-holding major elements other than calcium from the half-strength nutrient solution. For the nutrient solution with cal-cium at 100 jug/g, the withholding of potassium, magnesium,sulfur, nitrogen, or phosphorus caused little or no effect onthe lengths of hypocotyls as compared with withholding ofcalcium (cf. Table III and Fig. 3). However, some treatments(in particular those supplied with nutrient minus magnesiumor nitrogen), produced a few seedlings which developed hy-pocotyl collapse symptoms. Since the level of calcium in thenutrient solution was close to the minimum required for growthof normal seedlings (Fig. 3), this development of symptomscould have been due to reduced uptake or utilization of cal-cium in the absence of the deficient element. This possibilitywas tested in two further experiments in which the level of cal-cium was raised to 200 ,ug/g while the levels of other elementsremained as in the previous experiments. Under these con-ditions, only one plant developed symptoms of hypocotylcollapse (Table III). The data are in accord with the conceptthat, when the level of calcium is marginal for normal growth,the withholding of certain elements, e.g., magnesium or nitro-gen from a nutrient solution can induce calcium deficiencysymptoms.

Effect of Growth Medium on Hypocotyl or Epicotyl De-velopment in Several Legumes. Growth of five different cul-tivars of Phaseolus vulgaris (including cv. Pinto), one of Sojamax Piper, and one each of Vicia faba L. and Pisum sativumL. was examined under nonsterile conditions in perlite wateredwith (a) complete nutrient or demineralized water and (b) withhalf-strength nutrient or half-strength nutrient minus calcium.For the genus Phaseolus, hypocotyl collapse occurred in someplants of all cultivars receiving demineralized water or half-sterength nutrient minus calcium (Table IV). Symptoms did notdevelop when seedlings were grown in complete nutrient.Fewer plants developed symptoms when they received half-strength nutrient minus calcium than when they received de-mineralized water. The collapse symptom did not occur inSoja, Vicia, or Pisum.

For cultivars of Phaseolus and Soja the reduction in lengthsof hypocotyls relative to those of controls was greater whenseedlings were watered with demineralized water than whenthey were watered with half-strength nutrient minus calcium.The reduction in length of epicotyls of Vicia was about thesame for the two treatments. No reduction in epicotyl lengthwas observed for Pisum. The data showed that elongation ofhypocotyls or epicotyls of all the legumes, except Pisum, in-creased when an external supply of calcium was provided. Thegreater incidence of the hypocotyl collapse symptom amongplants grown with demineralized water than with completenutrient minus calcium could be due to the enhancement ofcalcium deficiency in the absence of other essential elements, assuggested by data in Table III, and also to the leaching ofessential nutrients from the seedlings into the demineralizedwater (15).The percentage of calcium in seed of cutivars of Phaseolus

vulgaris (Table V) was within the range of that for seed offield bean (Phaseolus vulgaris L.) cited by Beeson (3). Also, inagreement with his data, levels of calcium were lower thanthose of magnesium and potassium. For plants grown innutrient minus calcium, except in respect to Soja max, therewas no clear correlation between low incidence of hypocotyl

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CALCIUM DEFICIENCY OF PHASEOLUS SEEDLINGS

Table IV. Effect of Growth Medium on Growth of Hypocotyls and Epicotyls of Several Legumes'

Plaseolus vulgariss iaMedium' ____-____ ___ Soja max3 VColesfabar p.SMSjjUn

Pintouf| Brown Butter Red Red Lincoln Prolific Canner 75Totalno.ofpiPinto | Beauty Bean Mexican Kidney L C D

Total no.ofplantswithcollapsesymp- A 17 17 19 11 19 0 0 0toms B 0 0 0 0 0 0 0 0

Mean reduction in length of hypocotyls 79 62 74 62 78 41 35 0or epicotyls of A relative to B (%)

Total no. of plants with collapse symp- C 11 2 4 1 6: 0 0 0toms D 0 0 0 1 0 0 0 0

Mean reduction in length of hypocotyls 58 25 20 36 44 16 33 0or epicotyls of C relative to D(%) l_l_l_l

l Data for 20 seedlings per treatment (two experiments) grown under nonsterile conditions in perlite.2 A: plants watered with demineralized water; B: plants watered with complete nutrient; C: plants watered with half-strength nutrient

minus calcium; D: plants watered with half-strength nutrient.I Hypocotyls4 Epicotyls.

collapse symptoms and high level of calcium in the seed (cf.Tables IV and V).

DISCUSSION

Data in this paper show that calcium deficiency causes hypo-cotyl collapse symptoms of bean seedlings grown in the dark.Incidence of seedlings with this symptom decreased with in-crease in the level of calcium supplied to seedlings grownunder sterile conditions and was not directly associated withdificiencies of other major elements. Since neither fungi norbacteria could be isolated from collapsed hypocotyls of seed-lings grown under sterile conditions, the disease almost cer-tainly was not associated with a seed-borne pathogen. Data tobe presented elsewhere will show that the collapse symptomalso can occur in calcium-deficient bean seedlings grown in thelight. The symptom is comparable with the wilting observed inpetioles, peduncles, and stems of some plants grown undercalcium-dificient conditions in the light (9).

Relatively little is known about the role of calcium in shootgrowth, but as in root growth, it is thought to cause "stabiliza-tion of the wall during the second phase of the cell elongationinvolving a deposition of new wall material" (5). Studies on theshoot apex of barley (1 1) indicated that calcium is essential forthe maintenance of structural integrity of cll membranes. Theauthor suggested that the effects of calcium on the cell wallwere secondary to these changes. In the present experimentscalcium supplied in the external medium maintained hypocotylcell wall structure and promoted elongation of hypocotyls. Itpromoted elongation of the epicotyl of Vicia faba but not thatof Pisum sativum. The latter finding agrees with earlier work ofBurstrom (5), who found that, whereas the first internode ofpea seedlings grown in a nutrient solution deficient in calciumwas normal, the second internode was retarded.The minimal external level of calcium required for normal

hypocotyl growth closely approached 100 ytg/g (Fig. 3). Alower level of calcium may have been adequate in a nutrientsolution containing all essential ions at a lower balanced level(10).The present data suggest that the supply of calcium in the

cotyledons is either too small or insufficiently available to sup-port the rapid growth of the hypocotyl. The second alternativeseems more likely, since an increase in total calcium content inthe seed with increase in seed weight up to 0.45 g neither re-duced the incidence of the hypocotyl collapse symptom nor in-creased the lengths of hypocotyls (Table II). This apparently

Table V. Levels of Calcium, Magniesium, anid Potassiuim in Seed ofSeveral Legumes

Mean data for 10 seeds for cv. Pinto, 4 seeds for Soja and Pisum,and 2 seeds for other legumes.

Ca Mg K

% dry w

Phaseolus vulgaris cv.Pinto 0.109 0.158 1.24Brown Beauty 0.124 0.165 1.14Butter Bean I 0.139 0.157 1.12Red Mexican 0.124 0.174 1.11Red Kidney 0.067 0.154 1.21

Soja max cv. Lincoln 0.362 0.381 1.570Vicia faba cv. Coles Dwarf Prolific 0.123 0.142 0.795Pisum sativum cv. Canner 75 0.121 0.161 0.794

inadequate translocation of calcium from the seed to the rap-idly developing hypocotyl can be compared with the wellknown inadequate translocation of calcium from relatively ma-ture plant parts to rapidly developing seeds and fruits. A no-table example is the development of blossom end rot in a rap-idly developing tomato fruit (9, 13).

Acknowledgments-The author would like to thank Dr. D. Bouma of the Di-vision of Plant Industry, CSIRO, Canberra, for helpful discussion and ProfessorH. Mohr of the Biological Institute of the University of Freiburg for his interestin the project and for making available laboratory facilities. She also would liketo thank Professor G. Drews of the University of Freiburg and Dr. J. F. Brad-bury of the Mycological Instiute, Kew, London, for isolating and identifying,respectively, Pseudomonas testosteroni (MIarcus and Talalay), and Mrs. A.Andruska for efficient technical assistance.

LITERATURE CITED

1. ALBRECHT, W. A. AND H. JENN-Y. 1931. Avaliable soil calcium in relation to"damping off" of soy bean seedlings. Bot. Gaz. 92: 263-278.

2. ANGELL, H. R. 1950. Seedling blight II. Soil in relation to seedling blight ofopium poppy and peas. Aust. J. Agr. Sci. 1: 132-140.

3. BEESON, K. C. 1941. The mineral composition of crops with particular refer-ence to the soils in which they were grown. Misc. Pub. No. 369. UnitedStates Department of Agriculture, Washington, D.C.

4. BURSTROM, H. 1963. Gro-th regulation by metals and chelates. Advan. Bot.Res. 1: 73-100.

5. BURSTROM, H. G. 1964. Calcium, water conditions, and growth of pea seedlingstems. Physiol. Plant. 17: 207-219.

6. CLARK, B. E. AND D. B. KLINE. 1965. Effect of water temperature, seedmoisture content, mechanical injury, and calcium nitrate solution on thegermination of snap bean seeds in laboratory germination tests. Proc. Ass.Seed Anal. 55: 110-120.

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7. DAVID, D. J. 1959. Determination of calcium in plant material by atomicabsorption spectrophotometry. Analyst 84: 536-545.

8. GEORGE, C. F. 1953. Glossary. In: Plant Diseases: The Yearbook of Agri-culture. United States Department of Agriculture, Washington, D.C. pp.897-908.

9. HEWIrr, E. J. 1963. The essential nutrient elements; requirements and inter-actions in plants. In: F. C. Steward, ed., Plant Physiology, Vol. 3. AcademicPress, New York. pp. 137-360.

10. JONES, R. G. W., AND 0. R. LU-NT. 1967. The function of calcium in plants.Bot. Rev. 33: 407-426.

11. MARINos, N. G. 1962. Studies on submicroscopic aspects of mineral de-ficiencies. I. Calcium deficiency in the shoot apex of barley. Amer. J. Bot.49: 834-841.

12. McNEw, G. L. 1953. The effects of soil fertilitv. In: Plant Diseases: The Year

Book of Agriculture. United States Departments of Agriculture, Washington,D.C. pp. 100-114.

13. SPuRn, A. R. 1958. Anatomical aspects of blossom-end rot in the tomato withspecial reference to calcium nutrition. Hilgardia 28: 269-295.

14. STANIER, R. Y., N. J. PALLERONI, AND M. DOUDOROFF. 1966. The aerobicPseudomonads: A taxonomic study. J. Gen. Microbiol. 43: 159-271.

15. TRIUE, R. H. 1914. The harmful action of distilled water. Amer. J. Bot. 1:255-273.

16. WALKER, J. C. 1952. Diseases of Vegetable Crops. McGraw-Hill BookCompany, 'New York.

17. WILLIANIS, F. J. 1965. A hypocotyl collar rot of Phaseolus vulgaris. Plant Dis.Rep. 49: 134.

18. WILLIAMS, F. J., W. L. HOLLIS, AND M. H. DAY. 1966. Incidence of hypo-cotyl collar rot of Phaseoluts vulgaris in the field and in germination tests.Phytopathology 56: 531-535.

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calcium deficiency dark-grown seedlings phaseolus lof ph. control plants were grown in cotton wool...

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