ezekiel, fudge · strated that the growthandculture responses of these isolates were similar. the...

SOME FACTORS INFLUENCING THE UTILIZATION OF IN-ORGANIC NITROGEN BY THE ROOT ROT FUNGUS1

PAUL J. TALLEY AND LESTER M. BLANK

(WITH ONE FIGURE)

IntroductionThe utilization of different forms of nitrogen by Phymatotrichum omni-

vorum (SHEAR) DUGGAR has been studied by NEAL, WESTER, and GUNN(4), and by EZEKIEL, TAUBENHAUS, and FUDGE (3). The former used rela-tively high concentrations of ammonium nitrate and ammonium sulphatein an acid culture medium, and reported that salts containing the am-monium ion were poor sources of nitrogen and were even toxic to theorganism. They obtained fair growth with nitrates. EZEKIEL et al. reportedthat the organism used potassium nitrate, ammonium sulphate, ammoniumnitrate, and organic nitrogen. They obtained the best growth with am-monium nitrate. Potassium nitrate appeared to be a better source of nitro-gen than ammonium sulphate. There seems to be no basis for STEINBERG'Slisting P. omnivorum in group three (8) under the scheme of classificationemployed by ROBBINS (6), since this group includes those organisms capableof using only ammonium and organic nitrogeii. The work of EZEKIEL et at.and of NEAL et al. would place the root rot fungus in group two, which iscomprised of organisms able to obtain their nitrogen from nitrates, am-monium salts, or organic sources.

The absence of growth reported by NEAL et al. when ammonium com-pounds were added to the nutrient solution suggested to STREETS (9) theutilization of ammonium salts as a practical method for the control of theroot rot disease. The latter reported good results in controlling the diseasein this manner. Since these results appeared to be at variance with theexperiments reported by EZEKIEL et al. which indicated that ammoniumnitrate was an excellent source of nitrogen, it therefore seemed desirableto test thoroughly the relative nutritional values of the various inorganicforms of nitrogen. The present studies should answer some of the questionsconcerning the value of ammonium compounds as nutrients or as toxieagents for the root rot fungus.

Materials and methods

The cultural methods used in these studies were modifications of thoseemployed in earlier studies with this organism on the effect of the traceelements (1) and have been described in some detail in a study of the salt

1 Published with the approval of the Director as Technical Contribution no. 613 of theTexas Agricultural Experiment Station.

52

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

TALLEY AND BLANK: NITROGEN TTILIZATION BY ROOT ROT FUNGUS a3

requirements in synthetic nutrient solutions for the growth of this organism(10). The inoculum employed in the earlier experiments herein reportedwas from a culture of P. omnivorutm designated as isolate 28. In the laterexperiments isolate 53 was used. Routine tests were made which demon-strated that the growth and culture responses of these isolates were similar.The basic nutrient solution contained 0.008 M K2HPO4, 0.003 M MgSO4,0.002 M KCL, and additions of 2 p.p.m. each of Fe, Mn, and Zn. Glucosesupplied at the rate of 40 gm. per liter was the source of carbon throughoutthese studies. The variables in these experiments were limited to the sourcesand amounts of inorganic nitrogen unless otherwise indicated. All cultureswere incubated at 280 C. and the dry weights of the fungal mats wereadopted as the measures of growth and utilization of the various types ofnitrogen.

Experiments and results

A series of eleven different nutrient solutions, each containing the sameamount of nitrogen, was prepared. The first solution, which was the stand-ard for comparison throughout this series, contained 0.025 M NH4NO3.There were three different solutions containing only nitrate nitrogen. Theyconsisted of one with KNO3, another with Mg(NO3)2, and another with amixture of KNO3 and Mg(NOD)2 combined in such a way as to supply thedesired amount of nitrate nitrogen while maintaining the same K to Mgratio that existed in the basic solution. There were seven different nutrientsolutions containing all their nitrogen as ammonium. Three of these wereprepared by using NH4C1, (NH4) 3S04, or (NH4)2HPO4. Three more wereprepared by using these same ammonium salts in combinations of two 's. Ineach of these cases the proportion of the two ammonium salts was such as tomaintain the same ratios of their respective anions as existed in the originalbasic solution. The final ammonium solution was prepared by using allthree of these ammonium compounds in the correct proportions to main-tain the original ratio of P04: SO4: Cl occurring in the standard basic solu-tion. None of these eleven solutions introduced any new ions into theculture solution, excepting those introduced as impurities, and all containedthe same amount of nitrogen. The types and concentrations of the nitrog-enous salts used in these solutions are given in table I. Each solution wasprepared in 500-ml. portions and then divided into 50-ml. portions, auto-claved, and inoculated. The pH of each type of solution was measured afterautoclaving. All pH measurements were made with a pH meter equippedwith a glass electrode. Harvests of four flasks of each series were made onthe 18th and 24th day after inoculation. The mean weights of the dry matsand the range of the final pH measurements of the culture solutions aregiven in table I.


PLANT PHYSIOLOGY

TABLE IGROWTH OF P. omnivorum IN NUTRIENT SOLUTIONS WITH NITROGEN SUPPLIED BY VARIOUS

INORGANIC SALTS

NITROGEN1T SOURCES GROWTH RESPONSE GROWTH RESPONSE

NITROGEN______SOURCES PH AFTER 18 DAYS AFTER 24 DAYS

CNE- ORIGINALSALTS USED TI~TICON PH RANGE MEAN WT. PH RANGE MEAN WT.

M mg. mg.NH4NO3. 0.0250 6.8 4.3 - 4.7 665 4.9 -5.5 553KNO3 ... .... 0.0500 6.4 7.1-7.3 608 6.9 - 7.1 455Mg(NO3)2 ... 0.0250 6.1 7.2 - 7.5 424 7.0 - 7.1 739KNO3 + ........... 0.0364Mg (NO3)2 0.0068 6.5 7.4-7.6 777 7.3 -7.4 576

NH4C1 ... .... 0.0500 6.7 2.5-2.6 77 2.5-2.7 73(NH4) 2S04 ........... 0.0250 6.8 2.8 - 2.9 76 2.8 - 3.0 62

(NH4) 2HP04 0.0250 6.9 2.9-3.1 273 3.0 - 3.1 270NH4C1+ ........... 0.0125(NH4)2SO......4 0.0188 6.7 3.0 - 3.1 80 2.6 - 2.8 67

NH4C1+ .......... 0.0055(NH4) 2HP04 0.0222 6.7 3.0 -3.1 264 2.9 -3.0 258

(NE4) 2S04 + ...... 0.0068(NH4) 2HP04 0.0182 6.7 1.1-5.3 194 2.9 - 5.7 182

NH4C1 . ........... 0.0042(NH4)2S04 + 0.0063(NH4)2HP04 0.0167 6.9 3.0 - 3.1 190 2.7 - 3.0 177

The results show that the fungus can use ammonium or nitrate nitrogen.One would be justified in assuming that ammonium was a poor source ofnitrogen and that the nitrates were good, except for the lowering of the pHthat occurred with the small amount of growth with ammonium. In theculture flasks containing only ammonium nitrogen the pH dropped to 3.1or below. Only two individual cultures were exceptions to this very low pHat the time of harvest and they were accompanied by the smallest matweights. It could be assumed in these two cases that the higher pH valueswere either the result of a failure to grow rapidly or the results of an earlycessation of growth and accompanying autolysis. Regardless of theseexceptions, it is evident that growth results in a lowering of the pH whenammonium is the only source of nitrogen. This low pH value tends to sup-press the growth of P. omnivorum.

The ability of the organism to use nitrates cannot be doubted. The cul-tures growing in nitrate solutions had the expected increase in pH, which wasmost noticeable in the flasks producing the heaviest mats. The most rapidgrowth was produced on those solutions with an extra amount of potassium,but the heaviest mats were produced in solutions in which the basic balance

54


TALLEY AND BILANK: NITROGEN UTILIZATION BY ROOT ROT FUNGUS 55

between K and Mg was maintained. It is evident that the relative values ofammonium and nitrate nitrogen in the nutrition of the fungus cannot beascertained unless the divergent shifts in the pH values are controlled.

INFLUENCE OF CACO3 ON NITROGEN UTILIZATION

An experiment on the utilization of ammonium was conducted in whichthe seven types and combinations of ammonium salts listed in table Iserved as the sources of nitrogen. A nutrient solution containing 0.025 Mammonium nitrate again served as the control or basis of evaluation. Inthis experiment each nutrient solution was prepared in the proper amountto yield twenty portions of 50 ml. To ten of these portions enough calciumcarbonate was added to bring its concentration to 0.025 M. This amountof calcium carbonate should neutralize all of the acid that would be developedfrom any of the ammonium salts if all of their ammonium ions and noneof their anions were absorbed by the growing organism. The fungal matsfrom four cultures of each type of solution were harvested after 20 and 24days incubation. The results of this experiment are given in the lowerportion of table II.

While the growth in this experiment was poor, as indicated by thelight mats obtained from the control solution (NH4NO3), the results withthe different ammonium salts and their various combinations, when calciumcarbonate was omitted, gave the same general trend obtained in the pre-vious experiment. Growth on ammonium chloride, ammonium sulphate, andthe combination of the two salts was limited and was accompanied by amarked increase in acidity. Ammonium phosphate again was the best sourceof ammonium nitrogen. The combinations of ammonium phosphate with oneor both of the other ammonium salts were better than the sulphate or chlorideof ammonium used singly or in combination.

The addition of calcium carbonate to the various ammonium solutionsproduced a significant improvement in nitrogen relations as indicated bythe heavier mat weights. This was not evident in the ammonium nitratesolution but its pH did not change sufficiently to become a dominantfactor during growth when calcium carbonate was omitted. The beneficialaction of calcium carbonate is most noticeable with ammonium chloride, am-monium sulphate, and combinations containing one or more of these salts inappreciable quantities.

Another experiment was performed using potassium nitrate, magnesiumnitrate, and a mixture of the two. The amount and proportions of thesenitrates was the same as in the corresponding solution shown in table I.An additional series supplying the same amount of nitrogen in the form ofcalcium nitrate was included. The basic solution using ammonium nitratewas the standard control. All nutrient solutions were prepared with and


PLANT PIIYSIOLOGY

without 0.025 M caleium carbonate. The results are given in the upper partof table II. The weights of the mats produced in the solutions without Cal-cium carbonate confirm the results shown in table I. There was an increasein pH resulting from growth with nitrate nitrogen. This was most pro-nounced in the case of the solution containing calcium nitrate, which wasthe poorest of the four nitrate sources. The addition of calcium carbon-ate produced no beneficial changes in mat weights except in the solutionscontaining potassium nitrate alone or in combination. There appeared tobe some beneficial action of calcium carbonate in solutions in which thecontent of potassium was inereased. The poor growth with calcium nitrateindicates that calcium compounds were present in excess of the optimumamount.

INFLUENCE OF MGCO3 AND NA2CO, ON NITROGEN UTILIZATION

The general effect of CaCO3 on the utilization of ammonium niitrogenwas to increase the amount of growth and to prevent to a certain degreethe accompanying decrease in the pH of the nutrient solutions. Sincethere was an indication of some action of calcium carbonate which mightbe attributed to the effect of the calcium ion, it was considered advisableto repeat the experiments reported in table II, using magnesium carbonateand sodium carbonate instead of calcium carbonate. These experimentsgave some interesting effects which were ascribable to the increase ofmagnesium or to the addition of sodium.

The most noticeable action of 0.025 M magnesium carbonate was thecomplete suppression of growth in solutions containing nitrogen suppliedas ammonium nitrate, ammonium chloride, ammonium sulphate, or as amixture of ammonium chloride and sulphate. Growth was poor in thecase of magnesium nitrate and in the mixture of potassium nitrate and mag-nesium nitrate. There was a marked increase in growth in the solutionscontaining dibasic ammonium phosphate singly or in combination with theother ammonium salts. Growth with calcium nitrate was not materiallyNaffected, but was delayed slightly in the case of potassium nitrate.

The complete suppression of growth with the addition of 0.025 M mag-nesium carbonate to certain of the nutrient solutions and its contrastingbeneficial action on the others suggested some direct or indirect effect of themagnesium ion. It was considered desirable, therefore, to repeat theexperiment using a 0.0123 M concentration of magnesium carbonate insteadof the 0.025 M concentration of this salt.

The results of this experiment can be summarized by saying that theyconfirmed, in general, the results of the previous experiment. Magnesiumcarbonate inhibited all growth, in solutions containing ammonium nitrate,ammonium chloride, ammonium sulphate, or a mixture of ammonium chloride

56


TALLEY AND BLANK: NITROGEN UTILIZATION BY ROOT ROT FUNGUS 57

TABLE IIEFFECTS OF THE ADDITION OF 0.025 M CACO3 ON GROWTH OF P. omnivorum IN NUTRIENT

SOLUTIONS WITH NITROGEN SUPPLIED BY VARIOUS INORGANIC SALTS (NITROGENCONTENT EQUIVALENT TO 0.0250 M NH4NO3)

GROWTH RESPONSE GROWTH RESPONSE

NITROGEN CACO3 ORIGINAL AFTER 20 DAYS AFTER 24 DAYSSOURCE 0.025 M PH MEAN MEAN

PH RANGE WEIGHT PH RANGE WEIGHT

mg. mg.NH4NO3 _ 6.6 5.4- 6.1 607 5.6 - 6.0 622

+ 6.8 6.1 - 6.4 604 6.6 - 7.0 636

KNO3 .... 6.4 7.6 - 7.7 330 7.3 - 7.4 392+ 6.7 7.6 - 8.0 304 7.2 - 7.7 584

Mg(N03)2 ............ - 6.3 7.7-7.8 330 7.3 - 7.4 598+ 6.6 7.8 - 8.1 354 7.8 - 8.0 515

KNO3 +Mg(N03)2 _ 6.7 7.4-7.8 578 7.2 - 7.6 477

+ 6.9 7.8 - 8.0 484 7.6 - 7.9 774

Ca (NO3)2 ............... - .0 6.8 - 7.1 220 7.0 - 7.4 300+ 7.3 6.9 - 7.2 157 6.8 - 7.0 225

NH4NO3 - 6.9 6.3 - 6.6 199 6.4- 7.3 375+ 7.0 6.4 - 6.6 229 6.4 - 7.1 296

NH4C1 _ 6.7 3.0 - 3.1 61 2.8 - 3.0 60+ 6.9 4.9 - 5.3 242 4.5 - 4.8 306

(NH4)2SO4 - 6.8 3.1 - 3.2 66 3.0 - 3.2 67+ 6.9 5.4 - 5.5 189 5.1 - 5.3 259

(NH4) 2HP04 - 6.9 3.2 -6.5 89 3.1 - 3.2 252+ 6.9 5.2 - 6.0 175 4.7 - 5.0 333

NH4C1+(NH4)2SO4 _ 6.7 3.1 - 3.5 68 3.1 - 3.1 60

+ 6.9 4.5 - 5.3 260 4.3 - 5.2 393NH4C1 +(NH4)2HP0 - 6.9 3.2 - 4.1 200 3.0 - 3.2 235(NH4) 2HP04 + 6.8 5.3 - 5.8 181 5.1 - 5.2 318

(NH4)2SPO4 - 6.8 3.2 - 5.5 150 2.9 - 3.3 205(N4) 2HP04--- + 6.9 5.2 - 5.6 213 5.2 - 5.5 255

NH4C1 +(NH4) 2SO4+(NH4)2HPO04 - 6.9 3.1 - 4.8 158 3.0 - 3.1 178

+ 6.9 4.9 - 5.7 192 5.3 - 5.5 200

and ammonium sulphate. Very little growth occurred with magnesiumnitrate but the mixture of potassium nitrate and magnesium nitrate was un-

affected by this lower concentration of magnesium carbonate. Potassiumnitrate and calcium nitrate gave better growth when accompanied by mag-nesium carbonate. Dibasic ammonium phosphate and all combinations of


PLANT PHYSIOLOGY

ammonium salts in which high phosphate were present gave excellentgrowth when accompanied by magnesium carbonate.A similar experiment was conducted using 0.0063 M sodium carbonate

with the twelve nutrient solutions containing the different nitrogenous saltssingly and in combinations. The general effects of sodium carbonate weremore similar to those of calcium carbonate than to those of magnesiumcarbonate. It differed from the higher concentration of calcium carbonateby being more unfavorable with ammonium nitrate and with potassiumnitrate either singly or in combination with magnesium nitrate. It was lessinhibitory than calcium carbonate when used with calcium nitrate.

EFFECT OF CARBONATE WITH A LIMITED NITROGEN SUPPLY

In an earlier study it was found that the amount of growth dependslargely on the amount of glucose available, if nitrogen is not limiting,but that the rate of growth is determined largely by the amount of availablenitrogen, if the carbon is not limiting (10). It was therefore consideredadvisable to decrease the amount of nitrogen from the equivalent of 0.025 Mto 0.0125 M ammonium nitrate. This decrease in nitrogen content permitteda corresponding decrease in the amount of carbonate that would be requiredto offset the potential acidogenic effect of ammonium salts. These changestended to diminish the physiological importance of the accompanying in-crease in the concentrations of Ca, MIg, or Na ions supplied by the carbonatesand the Ca, Mg or K ions derived from the nitrates. It was assumed that thegeneral decrease in the concentration of the electrolytes and the resultingmodification in the ionic ratios, Mg: K, Mg: Ca, and Mg: K: Ca, would per-mit a more reliable evaluation of the effects of nitrate and ammonium nitro-gen in the nutrition and environment of the organism.

An experiment using the twelve nitrogen sources, listed in table II,was conducted in which the quantity of nitrogen was reduced to theequivalent of 0.0125 M ammonium nitrate and the effects of calcium car-bonate, magnesium carbonate, and sodium carbonate on each source of nitro-gen were studied simultaneously. This resulted in 48 different nutrientsolutions. Isolate 53 was used in this and subsequent experiments asthe source of inoculum. Five cultures of each type were harvested after20 days incubation. The results of this experiment are given in table III.The mean weight of the mats produced in the various solutions are presentedgraphically in figure 1.

The data on the various nitrogen sources without the addition of anycarbonates show that good growth occurred on the control, ammoniumnitrate, and all the various nitrates, singly and in combination. Goodgrowth with- nitrates as the source of nitrogen was accompanied by slightincreases in the pH of the culture solutions. In these solutions the pH

58


TALLEY AND BLANK: NITROGEN UTILIZATION BY ROOT' ROT FUNGUS 59

values did not become critical. The results with ammonium nitrogenshow that poor growth is accompanied by the usual increase in acidity.The ammonium salts of the strong acids were the poorest sources anddeveloped the greatest acidity. Increasing amounts of ammonium phos-phate resulted in increased weights. These results agree very closelywith those shown in tables I and II.

The effects of calcium carbonate differed with the various nitrogen

500^ TYPE OF CARBONATE ADOED

4 NDE c,mcN,

I~~~~~~~V 200

00NH. NO, ONo" mg K - C~ )NO CIN H.IC0, (NlI)ROP. H.CI &.CI N,aN9g 00,) (NR,),OO, (I.PO. ORHO,4PO. (NH,),5R,

DIFFERENr sOOEI oF E0VAI TNT AWXWI!N OF NITRGEN

FIG. 1. The growth of P. omnivorum when the nitrogen is supplied by differentammonium and nitrate salts with and without the addition of CaCO3, MgCO2, or Na2CO3.

sources. There was not as much modification of the amount of growth asin the earlier experiments where the concentrations of calcium carbonateand the nitrogenous salts were twice as high. There was no significantchange with ammonium nitrate. Growth in solutions containing potassiumnitrate was slightly improved by the addition of calcium carbonate whilethat in solutions containing magnesium nitrate was impaired. These resultsagree with those presented in table II. In the earlier experiments theaddition of calcium carbonate improved growth in the mixture of potassiumnitrate and magnesium nitrate, but with the lower concentration of calciumcarbonate employed in this experiment no improvement was observed.

All of the cultures containing their nitrogen as ammonium were greatlyimproved by the addition of calcium carbonate. This was most noticeablein the solutions with additional amounts of sulphates and chlorides. ThepH was lowered by growth but did not become limiting. All of the am-monium solutions produced good fungal mats when calcium carbonate waspresent.

The effects of magnesium carbonate can be compared to those describedin the earlier experiments in which nitrogen was twice as abundant.


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Magnesium carbonate did not impair growth in the ammonium nitratesolution but did in all other nitrate solutions with the single exception ofcalcium nitrate. The pH values after 20 days incubation were the highestwith the nitrate sources to which magnesium carbonate was added.

The effects of magnesium carbonate on the utilization of ammoniumwere of two general types. When nitrogen was supplied as ammoniumphosphate, alone or in combination with other ammonium salts, goodgrowth resulted. The best growth obtained from any of the ammoniacalsolutions occurred in those containing an increased amount of phosphatesplus magnesium carbonate. When phosphates were not increased the utiliza-tion of ammonium in the presence of magnesium carbonate was poor.

In general, the effects of sodium carbonate on the utilization of ammoniumwere similar to those of calcium carbonate. It was a little more effectivethan calcium carbonate in retarding, the development of acidity and pro-duced heavier mats in most of the solutions containing high concentrationsof sulphate. It was slightly less beneficial than calcium carbonate in theother solutions containing ammnoiium.

In the nitrate solutions sodium carbonate differed from calcium carbo-nate by being less favorable with potassium nitrate and the mixture ofpotassium nitrate and magnesium nitrate, and by being more favorable withcalcium nitrate.

UTILIZATION OF NITRATE NITROGEN

After demonstrating that nitrate and ammonium nitrogen are of essen-tially equal value for the growth of the fungus in properly adjusted nutrientsolutions, it seemed desirable to determine the ability of the organism touse nitrite nitrogen. Potassium nitrite was used to supply the nitritenitrogen. Ammonium nitrite and ammonium niitrate were used as controlsolutions. Cultures containiing each of these three nitrogen compoundswere prepared in which the total nitrogen content of each individual cul-ture flask was the equivalent of that in the 50-ml. portions of the 0.0125 Mammoniuni nitrate control. Since nitrites are less stable chemically thannitrates or ammonium the proper amounts of KNO2 or NH4NO2 were intro-

TABLE IVGROWTH OF P. omjinivorum WITH NITRATE NITROGEN

NITROGEN SOURCE ORIGINAL PH PH RANGE AT MEAN WEIGHTS

SALT USED CONCENTRATION

M1l mg.NH4NO3 ...... 0.0125 6.7 4.7-5.6 598NH4NO *----- 0.0125 6.3 6.7-6.8 264KNO2 ............ 0.0250 6.3 7.2-7.3 175


PLANT PHYSIOLOGY

duced into dry sterile flasks as concentrated aqueous solution. Ethyl alcoholwas then added to assure sterilization. The solvent was allowed to com-pletely evaporate from the plugged flasks before a 50-ml. portion of asterilized solution containing the basic salts and glucose was addedaseptically. The inoculated flasks were incubated for 20 days. Theresults, based on five cultures of each type, are presented in table IV.

DiscussionThe amounts of growth obtained in nitrate and in ammonium cultures

are largely the result of the acidophobic nature of the fungus, the tendencyfor the ammonium cultures to become acid following the absorption of theammonium ion from the solution, and the reverse tendency in the solutionscontaining only nitrate nitrogen. These fundamental differences are clearlyillustrated in tables I, II, and III. The salt or salts used to supply thedesired form and amount of nitrogen and the addition of different carbonateshave some basic effects on the properties of the solutions and their physiol-ogical adaptability for growth of the organism. Some of these effects will bediscussed under separate headings.

GENERAL EFFECT OF CARBONATES

The tendency of cultures containing ammonium ions to become acidmasks the full nutritive value of this form of nitrogen. The addition ofcarbonates prevents the development of critical acidity in the ammoniumsolutions (tables II and III). Under these conditions ammonium becomesa favorable source of nitrogen (fig. 1, tables II, and III). The addition ofvarious carbonates to the different ammonium solutions does not produce anequal or proportionate effect in all cases. This is presumably connected withbasic modifications in the availabilities of certain essential ions in thenutrient solution. There is also some indication of an influence of these ionson nitrogen utilization.

The addition of carbonates to the various types and combinations ofnitrate salts produces variable effects. Unless the balance of certainmajor cations is materially improved by the cation accompanying thecarbonate, there is no consistent beneficial effect to be obtained by theaddition of carbonates to solutions supplying nitrogen in the nitrate form.In many cases there is a noticeable decrease in the growth.

AVAILABILITY OF TRACE ELEMENTS

There are several theoretical effects resulting from the additions of thethree different types of carbonates other than their common ability to retardthe development of acidity. When carbonates are added to nutrient solu-tions in concentrations as high as those employed in these experiments

62



(0.0125, anid 0.025 M) they will precipitate, or binid, the trace elements suchas iroln, imianganese, and zinc (2, 5, 7), which are required for the growth ofthis organism (1). If the reaction of the medium remained approximatelvneutral, or became more alkaline, these essential ions would have theiravailability decreased. This effect is probably more important in thesolutions conitaininig only n-itrate nlitrogen, since their reactions do notprogress toward acidity, than it is in the ammonium solutions. The reducedavailability of the trace elements probably- accounits for a part of the poorergrowth with eertaini nitrates wheni carbonates are added.

EFFECTS OF CATIONS ON NITRATE UTILIZATION

Aniother effect of the additioni of carboniates is the change in the absoluteamounits of the several major cations anid the resulting changes in proportionsor balances existing between them. The additioni of different kinids of carbo-nates would therefore be expected to produce different effects with thedifferent sources of nitrogen employed in the various solutions. The im-portaniee of the balanee between the major cations should be most evident

TABLE VINFLIUENCE OF THE CONCENTRATION OF AND THE BALANCE BETWEEN K AND MG ON GROWTH

OF P. onnivorimi WITHI TWO CONCENTRATIONS OF NITRATE NITROGEN

SOUTRCE OF CONCEN- CONCEN- CONCEN- K TO MG RELATIVENITRATE TRATION TRATION TRATION

RATIO VALUEoF NO, oF K OF MG VALUE

'in. iilol 111. i1ot 'In. molKNOS *.............................. 50 68.0 3.0 22.60 26.7*Mg (N°3)2 .............. 50 18.0 28.0 0.64 26.7*KNO3 + Mg (N03) 2 50 54.4 9.8 5.60 46.7*

KNO3.-..---....-----. 50 68.0 28.0 2.43 54.3 tMg(NO3)2 .................. 50 18.0 43.0 0.42 13.0 tKNO3 +Mg(NO3)2 50 54.4 34.8 1.56 32.6t

KNO3. ............ 50 68.0 15.5 4.39 34.0 tMg(NO3)2 5.................60 18.0 40.5 0.44 12.7 +

KNO3+ Mg (NO) 2 50 54.5 22.3 2.44 53.3f+

KNO3 ............. 25 43.0 3.0 14.33 18.7SMg (NO3) 2 25 18.0 15.5 1.16 23.6§KNO3 + Mg(N0O3)2 25 36.2 6.2 5.84 22.8§KNO3. ............ 25 43.0 15.5 2.77 12.6§Mg (NO3) 2 .................. 25 18.0 28.0 0.64 4.9§KNO3+ Mg(NO)2 25 36.2 18.7 1.94 17.4§

* Relative growth values based on mean weights shown in table II under 20-day har-vest; nitrate solutions lacking Ca were the only ones supplying applicable data.

t, + Growth values calculated from experiments not reported elsewhere in this paper.Solutions designated (t) had 0.025 M MgCO3 while those designated (+) had 0.0125 MMgCO3 additions.

§ Values obtained from data reported in table III (nitrate solutions without car.bonate and with MgCO3).


PLANT PIIYSIOLOGY

in the nitrate solutionis sinee the basic proportion-s of the essential magfnesium-ianld potassium ionls vary the mllost in them.

In table 1r the results of (liffereilt concentrations of potassium nitrate,magnesium niitrate, anid the balanced mixture of potassium nitrate andimnagnesium nitrate with and without the addition of 0.125 AI or 0.023 Almagnesium carbonate are assembled. This facilitates a studly of the effectof the balan-ice betweeni the potassium-l anid mao-nesium iolns onl growth with twodifferent concentrations of niitrate nitrooYen. The amounts of the essentialcatiolis, with the exception of K and AMg, are held constaiit in all thesesolution-s except for the slight deviations resultinig from the impurities ofthe reag,enits. The table inlcludes data from four different experimentstwo of which are described unlider results. The coimiparative effects of thebalanee between potassium amidmeagn(.esium cannot be valid between differ-ent experimients sinice the vigor of the fulngus varied from time to time anddifferent isolates were used. The "'relativ\e growth value" of any particularsolution is a percentage value obtaine(d bv dividing the mean weig'ht of thefungal mats produeced on it by the suiiimmation of the meani weights of thethree, or more, solutions from that experiment supplying (lata applicable tothe point under consideration. The "'relativ-e growtl value" permits a directcomparisons between experimenits.

With the higher coneenitrationis of nitrate nitrogen the best miiol frac-tionial ratios of potassium to magnesium are between 2.0 and 5.6. Therelative growth values are low for the solutions having a K: Mg ratio above5.6 or below 2.43. This range is broad considerinig the extremely high con-centrations of the potassiumn and miagnesium ions present in some of the solui-tions. The data con-cerninlg the K to AMg balanee when the lowver concenitra-tion of nitrate nitrogen is use(l in(licate a broader range of tolerance. Theextreinely highl concentrationis of either K or Mo are lacking in these soluitionsand the importanee of antagoonism is diminished accordingly. Under theseconditions good relative growth values accompany K to MIg ratios rangingfrom 1.16 to 14.33.

It is evideent from this analysis that the fungus, when growing onnitrates, has a wide range of tolerance to K or Mg if the ratio between themis not extremely high or low. This effect is similar to the interactions ofMg7SO4 and K2HPO4 reported in an earlier study (10).

The introduetion of additional cations as calcium earbonate or sodiumlicarbonate anld by the use of calcium nitrate produce some modifications in theinteraction of K and Mg. Some data pertainingy to these modifications areassembled in table VI. The content of nitrate, sulphate, phosphate, andchloride is held constant. The only variables are the cations listed in thetable. Since all of these solutions are parts of one experiment, which was

64


TALLEY AND BLANK: NITROGEN UTILIZATION BY ROOT ROr1 FUNGUS 65

presented in table III, direct comparisons betwveen any two solutions arevalid.

TABLE VIEFFECTS OF THE BALAN-CE BETWEEF,x K, MG, CA, AND NA ON THE GROWTH OF P. omnivorun

IN SOLUTIONS CONTAINING A LIMITED AMOUNT OF NITRATE NITROGEN(NITROGEN CONTENT EQUIVALENT TO 0.025 MI KNO3)

CONCENTRATION OF MAJOR CATIONS RATIO OF RELATIVERATIo OF GROWTH

K M,Nlg Ca Na VALUES*

71.rnol 71.rnol m'.mol m.mnol43.0 3.0 ...... ...... 14.33 7.0043.0 15.5 ...... ...... 2.77 4.7343.0 3.0 12.5 ...... 14.33 7.7843.0 3.0 ...... 25 14.33 5.7118.0 15.5 ...... ...... 1.16 8.8518.0 28.0 ...... ...... 0.64 1.8418.0 15.5 12.5 1.16 6.7318.0 15.5 25 1.16 6.6736.2 6.2 ...... ...... 5.84 8.5336.2 18.7 1.94 6.5136.2 6.2 12.5 ...... 5.84 8.3536.2 6.2 25 5.84 6.4618.0 3.0 12.5 6.00 6.2518.0 15.5 12.5 1.16 6.3018.0 3.0 25.0 6.00 2.8718.0 3.0 12.5 25 6.00 5.41

* All relative growth Values calculated from the data pertaining to the Various typesof nitrate solutions in table III, -with no carbonate additioni and with the addition ofMgCO, CaCO3, or Na2CO3.

The effect of calcium is interesting. A high K to Mg ratio, such as 14.33,is improved by the addition of Ca, while a low ratio, 1.16, is impaired bythe addition of Ca. Intermediate ratios are not noticeably affected by theaddition of smiiall ailmounts of Ca but are less favorable if large amounts of Caare added. Calciumi has a general effect comparable to the addition of asimilar amount of magnesium. This does not excludle the possibility of someindividual and specific action of the Ca ioni.

The effeet of Na is siilnilar to that of potassium. The addition of sodiumimpairs growth if the K to MIg ratio is high but it is beneficial if the ratiois low.

These studies show that the source of nitrate is of importallce onlyinsofar as the accompanyving cations are active in inifluencing its availabilityor utilization and the general reaction of the fungus to the solution.

EEFFECT OF VARIOUS IONS ON AMMONIUM UTILIZATION

The maliner in which these experiments were conducted offers limitedopportunities to observe the influence of the K to Mg ratio on the utilization


PLANT PhIYSIOLOGY

of amimonium. The basic K to Mg ratio results from the noni-nitrogenoussalts ancd prevails ini all the solutions to which magnlesiuin carbonate is notadded (fig. 1, table III). The basic ratio of 18: 3 is chaniged to 18: 15.5 bythe addition of magnesium carbonate. Both of these ratios are in the rangepermiiittinlg good growth. The ammonium solutions, however, afford theoportunity of studying the effects of the mlajor anionis, Cl, SO4 anld PO4singly and in combiniation. The main effect of the carbonate radical isassumed to be connected primarily with the preventioni of active acidity.If this is the ease, its concenltration would not remain conistant and anyconclusion regarding its effects would be questionable.

The sulphate ion, which is essential, produces an interestinig effect inthese solutions. The growth of the funigus tends to decrease with inereasingconcentrations of this radical. This is very noticeable when magnesium isincreased but is less critical when calcium or sodium is added to the solution.This effect of SO4 is most noticeable when all of the ammonium is suppliedas anmmoniumn sulphate. Growth is preven-ted in this type of solution whenmagnesium carbonate is added, eveen though the acidity is regulated. Withthe addition of ealcium carbonate, or sodium carbonate, good growth results.

There is a similar but less striking differenee between the interaction ofchlorides witlh calcium or sodium, and their interaction with magnesium.This less important effect of the chloride ion is presumed to be coiinected withthe high chloride toleranee of the funLus and its minimal requirement or

utilization of this particular ion (10).The addition of increasing amounts of P04 presents a different type of

effect. The lower solubility an-d the weak dissociationi of phosphate saltsalong with their beneficial buffer action may coiitribute materially to thisdifference. The ammonium phosphate solution and ammonium solutionscontaining inereased amoun-ts of phosphates are good sources of nitrogeni(tables II and III, fig. 1). If magnesium earbonate is added, ammoniiumsolutions with inereased amounts of phosphates produce fungal mats equalto or heavier than those produced by any other type of solution supplyingthe same amount of nitrogen as ammonium or n-itrate. The addition ofcalcium or sodium, while permitting good growth, does not produce thehighly beneficial effect of magnesium with these highli phosphate solutionls.The striking coninections betweeni high MIg and high P04 concentrations, inspite of the lowered and usually less favorable K to Mgu ratio, shows thatgrowth with ammonium nitrooen can be distinctly improved by simul-taneously inereasing the coneentrations of magnesium and phosphate.

No physiological explanation of the interaction of MIg and PO4 on am-monium utilization is offered. The roles of both of these ions in nutritionare numerous and involved. In this connection it should be mentioned thatprevious studies have showni that varying the basic solutioni by inereasing

66



the concentrationi of either AMg or PO4 does not materially imiiprove g,rowthof the fungcus when nitrogen is supplied as ammoni ium nitrate. There is,however, a significant and favorable effect associated with inereasing miiag-nesium sulphate and dibasic potassium phosphate simultanieously over awide ranae wbhen ammonium nitrate is the source of nitrogeni (10).

UTILIZATION OF NITRITE NNITROGEN

The utilization of nitrite nitrogeni in the case of solutions containingammoniumii nitrite cannot be assumed silnce the amount of growth obtainedis no greater than that which woould normally be produced by the ammoniumnitrogeni supplied by- this salt (table IV). It is evideent, however, that niitritenitrogen is not nioticeably toxic to this fungus. The growth of the fungcuson KNO2 supports the above conclusion regarding the toleraniee of thefungus for nitrites anid also shows that the fungus cani use nitrite nitrogenin its metabolismii ancd growth.

Summary

Experimlenits were coniducted on the growth of P. omm iomo)TI) in artificialnutrient solutions using various inorganiie salts as the sources of nitrogen.The utilization of ammonium, nitrate, aiid nitrite nitrogen was studied.

The growth of the fungus, when ammonoiumi is the only source of nitrogen,results in the developmenit of acidity in the culture solution while nlitrateutilization results in the developmenit of a more alkaline reaction.

The addition of ealcium carbonate, miao-nesiinm carboniate, or sodiumcarbonate to the nuiitrient imiedium permits good ogrowth with ammnnoniumnitro"en by delayinig the developmenit of critical acidity.

There is no apparent toxicity of ammonium, per se, in a properly7 coni-stituted anid balaneed nutrient solution. If these conditions are met, am-monium is an excellent source of nitrogeni.

Growth with niitrate nitrogen is influenced bv the balance betweenK and AMg. Calciumii ean be suLbstituted for Mg to a limilited extenit. Sodiumcan partially replace K in this anitagonistic actionl.

Ammoniiumio utilization is inifluenieed byN the ionic balance in the solution.There are signlificanit and favorable g-rowth reactionis with high Mg anid highP04 and with hiih Ca or Na and high SO4 or Cl content omi the utilizationof ammnnoniutm. There is a conitrastingo mifavorable growth reaction withhigh MIg when accompaniied by high 804 or Cl content.

The funglus cani grow wbhen all its niitrogen is supplied in the nitrite formii.No evidenee of niitrite toxicity was fouind.

TEXAS AGRICULTURAL EXPERI]MENT STATION, AND

BUREAU OF PLAN-T IXNDUSTRY


PLANT PIIYSIOLOGY

LITERATURE CITED

1. BLANK, LESTER M. Response of Phlymszatotrichluuxn Oflait-orl(in to eertaintrace elements. Jour. Agr. Res. 62: 129-159. 1941.

2. BORTELS, H. i-ber die Bedeutulng Yon Eisen, Zink, und Kupfer furMIikroorganismen. Biochein. Zeitsch. 182: 301-358. 1927.

3. EZEKIEL, AVALTER N., TAUBENHAU-S, J. J., and FUJDGE, J. F. Nutritionalrequirements of the root-rot fungus, Phlymiatotrichlun 0omn1ivorum.Plant Phvsiol. 9: 187-216. 1934.

4. NEAL, DAVID C., WESTER, R. E., and GUNN, KENNETH C. Growth of thecottoni root-rot funigus, in synthetic media, ancd the toxic effect ofammonia on the fungus. Jour. Agr. Res. 47: 107-118. 1933.

5. ROBERG, M. igber die Wirkung von Eisen-, Zink-, und Kupfersalzenauf Aspergillen. Centralbl. Bakt. 2 (Abt.) 74: 330-370. 1928.

6. ROBBINS, W. J. The assimfilation by plants of various forni-s of niitroeln.Amer. Jour. Bot. 24: 243-250. 1937.

7. STEINBERG, ROBERT A. Nutrient solution purification for removal ofheavy metals in deficiency inivestigations with Aspergillts niger.Jour. Agr. Res. 51: 413-424. 1935.

8. . Growth of fungi in synthetic niutrielnt solutiolns. Bot.Rev. 5: 327-330. 1939.

9. STREETS, R. B. Phymatotricliunm (cottoil or Texas) root rot in Arizona.Arizona Aogr. Exp. Sta. Bull. 71: 299-410. 1937.

10. TALLEY, PAUL J., and BLANK, LESTER M. A critical studiy of the niutri-tionial requirements of P y)nmatotrichurtmwimii?voriumi. Plant Phvsiol.16: 1-18. 1941.

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ezekiel, fudge · strated that the growthandculture responses of these isolates were similar. the...

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