THE INCIDENCE AND ENZYMATIC ACTIVITY OF MOLDS FOUND IN INDIANA AND OHIO TOMATOES Thesis Ior II" Dunn of pIm. D. MICHIGAN STATE UNIVERSITY Lawrence Sinclair White 1957 «r9 if.‘ ‘5" I'- This is to certify that the thesis entitled presented by LC.‘.-'1LEC'IIB SI'ICLAIK KTITE has been accepted towards fulfillment of the requirements for Mdegree inflifltflhiflogjf & P. Health f/u @/ Major professor Date QM 7 l / ?°57 / / f ’ 0-169 LIBRARY Michigan State University ‘. 'nsal I J: I, :51qu t t THE INCIDENCE AND ENZYMATIC ACTIVITY OF MOLDS FOUND IN INDIANA AND OHIO TOMATOES By Lawrence Sinclair White AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health Year 1957 Approved by SE:;au§ngz§ZD¢3E£€EEZLL¢436E3\\ ABSTRACT I This study was undertaken to determine the genera and species of fungi present in trmatoes and the ability of these molds to produce enzymatic and other changes in tomato fruits. It was thought that the pectolytic enzyme polygalacturonase (PG) might be of more significance than generally has been realized. Also of importance was the relation of the amount of visible root and mold growths to mold counts. Mold counts have been used as a criterion of the quality of tomato products since 1911., Tomatoes showing mold growths were collected from a wide- spread growing area in the states of Indiana and Ohio. The genera and species of molds present were correlated with the gross deterioration evident in the tomatoes. The behavior of molds in producing flavor, odor and pH changes was deter—r mined. Subsequently sound whole tomatoes were inoculated with the molds and the symptoms were described. Howard mold counts were made on comminuted tomatoes which showed various fungus genera and known percentages of visible rot. Also the influence of trimming Operations on mold counts was studied. The amount of PG and the enzyme cellulase (Cx) produced by the molds in tomato extracts was determined employing the "cup-plate" method of Reid (1950) and Dingle, Reid and Solo- mon (1953). Lawrence Sinclair White 2 Under field and experimental laboratory conditions the genera and species of molds which were present were of prime importance in determining the presence or absence of rot in tomatoes. Two of the most widely distributed molds in tomatoes, Alternaria solani and Colletotrichum phomoides often produced only minor lesions. By contrast, Oospora, Rhizopus, Fusarium and flgggg_sp. which generally do not appear as frequently under field conditions unless the humidity and temperature are high, were found to be the most active molds in producing rot. Toma- toes inoculated with ghizopus sp. showed the development of cracks. The genera and species of molds present influenced the pH and the flavor of juice extracted from the field tomatoes and similar changes were observed in inoculated tomato juice samples. While off-flavors usually were detected, some molds produced pleasant flavors and one strain of Penicillium sp. produced a flavor in tomato Juice which was preferred by tasters to that of uninoculated juice. In most instances Juice made from tomatoes which contained molds had high pH values and the examination of trims and culls showed appreciably higher pH values than sound whole tomatoes. The increase in the pH of tomatoes which contain molds was sug- gested as a factor which might contribute to the development 0f "flat sour" organisms and also subsequent spoilage of tomato Juice. “MU‘. vobvv ULva‘u‘L Juice made from tomatoes which had been trimmed invariably esrlcpwed higher mold counts than juice made from tomatoes which did not require trimming. High mold counts were found when very little rot was gazreesent, e.g., in one instance, as little as 0.1 percent xrj.s3ible rot by weight gave a Howard mold count of 50. While 31 senberg (1952b) made no mention of it, his data showed the same thing in several instances. These. data are in contrast 13:) the results of Howard and Stephenson (1917) which indicated “RDILd counts in excess of 50 percent positive fields were present orfily'when 5.5 percent or more visible rot by weight was present. Conversely, our work showed that a high percentage of visible rcrt nmy give low mold counts which was also previously shown (Iflisenberg, 1952a; Smith, 1952). The work reported here further demonstrated that there was not necessarily any correlation between the amount of visible rot and the Howard count since it is the type of mold which is the determining factor on the amount of visible rot present as well as having an important bearing on the mold count itself. In short, the type of mold which the Howard mold count does not and cannot determine is a more important con- sideration than the mere presence of infinitesimal mold frag- ments which may or may not be important from the standpoint of rot. Lawrence Sinclair White Ii The greatest PG activity was found in tomato tissue and in tomato juice samples which contained the most active rot- producing molds. In contrast to cellulase (Cx), the concen- trations of this enzyme closely paralleled the activity of the molds which produced the most rapid deterioration of tomato tissue. A considerable variation occurred in different strains of molds of the same species with respect to PG activity and amount of tissue breakdown in the tomato fruit. An outstand- ing example of this was found in Colletotrichum phomoides. fflais was a definite indication that the Howard mold count did not correlate with the amount of rot produced by some of the molds. In many incidences there was less rot produced by the same amount of growth of hyphae in some strains of the mold as shown by PG activity. __ Dingle, J., Reid, W. W., and Solomons, G. L. 1953. The enzy- Inatic degradation of pectin and other polysaccharides. - .Application of the "cup-plate" assay to the estimation of tenzymes. Journal of Scientific Food Agriculture, 3, 1h9-155. Eisenberg, W. V. 1952a. Factory control oftomato stock for ‘tomato products. Food Packer, 22(1), 38, no, h8, 68. ___ . 1952b. Observations and suggestions on factory control of rot and extraneous matter in tomato proaucts. iNational Canners Association, Washington, N.C.A. Informa- tion Letter 1371(36). Howard, B. J., and Stephenson, C. 1917. Microscopical studies on tomato products. United States Department of Agricul- ture, Bureau of Chemistry, Bulletin, 5§1, 1-2h. Reid, W. W. 1950. Estimation and separation of the pectin- ’iggerggg and polygalacturonase of micro-fungi. Nature, ‘9 0 - P fii' ‘ ‘4 ‘ THE INCIDENCE AND ENZYMATIC ACTIVITY OF MOLDS FOUND IN INDIANA AND OHIO TOMATOES ‘ ' By Lawrence Sinclair White A THESIS Silbmitted to the School for Advanced Graduate Studies of mehigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 TABLE OF CONTENTS Page LIST OF FIGURES ................................ v LIST OF TABLES ................................ vi ACKNOWLEDGMENTS ................................ vii INTRODUCTION ........................................ 1 PART I - STRUCTURAL CHANGES ASSOCIATED WITH THE PRESENCE OF MOI-IDS IN TOMATO FRUITS O0.0.0....OOOOOOOOCCOOOOOOOO Introduction I ................................... Review of Literature ........................... Experimental and Results ....................... Studies of Field Tomatoes .................... Laboratory Inoculation of Tomato Fruits with Malds oooeoeoeeeeeeeeeeeeeeeoeeeooooeeeeeeoee DiscuSSion ....OO...0....0.........O.............. PART 11 .. MOLD COUNTS, FLAVOR AND pH CHANGES IN RELATION TO STRAIN OF MOLD AND PERCENTAGE ROT PRESENT IN TOMATOES. I Introduction ..................................... REVIOW of Literature oeeoeooeeeeeeoeeeoeoeeeeeeee RelatiOnship between Mold Count and Percentage Visible Rot in Tomato Fruit ................. Influence of Mold Growth on Flavor, Color, and Odor of Tomato Juice ........................ Experimental and Results ........................ 11 A8 #9 TABLE OF CONTENTS (Cont.) PAGE Relationship between Mold Count of Tomatoes and Percentage Visible Rot................... h9 Mold Count of Trimmed Tomatoes Compared to Mold Count of Tomatoes Which Did Not Require Trimming eeeeooeeeeeooeeeeeeoeeeeeeoeeoooo 52 Influence of Factory Operations on hold Count and pH Of Tomato JUICO ooeeeooeooeeoeeeoeoe 55. Influence of Mold Growth on Flavor and pH 0f Tomato JUICQ eeoeoeeeoeeeoeeeeoooeooooee 58 D180u8810n .OOOOOOOOOOOOCOOOOOOOOOO...0.0.0.... 63 PART III - FUNGAL ENZYMES IN RELATION TO DECOMPOSITION OF TOMATO FRUITS .OOOOOOOOC.COOOCOOOOCOOOOOOOO0...... 65 IntrOdUCtion COCOCCOOOOOOCOOOOOOO0....0.00.... 66 Review of Literature .......................... 67 Role of the Pectic Substances in Plants .... 67 Chemical and Physical Characteristics of Tomtoea OOOOOOOOOOOOOOOOOOOOOOOO0.00.00... 68 Decomposition of Plant Tissues by Molds .... 71 Experimental and RESUItS eeeeeeeeeeoeeeeeeeeoee 77 Production of Pectinase (polygalacturonase) by the Molds as Determined by Liquefaction of. Pectate Gel .0000...OOOOOOOOOCOCOOOOOOOOOCO 77 Production of Polygalacturonase and Cellulase (Cx) by the Molds in Tomato Juice as Determined by Cup-Plate Assays oeeeeeeeoeeeeeeeeeeeeeo Polygalacturonase Concentrations in Partially and Wholly Decomposed Areas Trimmed from Tomatoes -.....0.00000000000000CCOO....0... 100 Discussion .0000...OOOOOOOOOOOOOOOOOOOOOO0.... 108 111 .3 #K '55.. . TABLE OF CONTENTS (Cont.) PAGE GENERAL DISCUSSION OOOOOOOOIOOOOOOOOOOOO00.00.000.00... 112 SWIM-RY 0.0.0.0...OOOOOOOOOOOOOOOOCOOOOO00.00.000.00... 118 BIBLIOGRAPI‘IY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO...0...... 121 iv TABLE OF CONTENTS (Cont.) PAGE GENERAL DISCUSSION OOOOOOOOOOOOOOOOOOOOO00.00.000.00... 112 SUM-IMY O...0.0.0.0000...OOOOOOOOOOOOOOO00.00.000.00... 118 BIBLIOGRAPIIY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0...... 121 iv HJRE L. LIST OF FIGURES Location of tomato sampling points in the states of Indiana and Ohio 0.0.0.0...OOOOOOOOOOOOOOOOOOO. ls Macroscopic characteristics produced on tomato fruits by some of the principal molds under field conditions 23 Effect of eXperimental inoculation of tomatoes with As or illus, Botr tis, Fusarium, Mucor, Oospora, hizoctonia, and Rhizopus ........................ 3h Effect of experimental inoculation of tomatoes with Alternaria, Colletotrichum, Hormodendrum, Penicillium and 13:6h0dorma 00000000000000.eeeeeeeeeeeeeeeeee 36 Pectate gel liquefaction experiments illustrated by A826r8111u8 Sp. and MUCCp EIObOSUB 0000000000000. 86 Comparison of the aerial mycelia produced by two strains of Fusarium oxysporium ..............,... 87 Differences in the growth characteristics of two strains of Alternaria solani cultured on pectate 861 medium OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 88 Differences in the growth characteristics of four strains of Colletotrichum phomoides cultured on pectate gel medium eeeeeeeeeeeeeeeeeeeeeeeeeeeeee 89 Modified Delineascope apparatus employed to magnify and project in the estimation of polygalacturonase activity in tomato extracts and cultures ........ 93 LDecreasing logarithmic concentrations of mold poly- alacturonase as shown by the cup-plate assay %P90t1n01 10M) eeeeeeeeeeeeeoeeeooooeeeeeeeoeoeee 95 Variation in polygalacturonase potency produced by 1h different m01d3 in tomato inice eeeeeeeeeeeeeeeee 96 Cup-plate zones magnified on grid showing measurable polygalacturonase activity in three tomato extracts, No measurable activity in one sample 1 l/u ....... 97 Variation in cellulase (Cx) potency produced by 1h different molds in tomato juice ................. 99 PAGE TABLE! III. III[. III. VII. VIII. VIII . IJE. XI. XII. XIII. LIST OF TABLES Symptoms Produced in Tomato Fruits when Mold Growth 18 Present 88 NOted by Various Workers eeeeeeeeeee Gross Characteristics of the Tomato Fruits Obtained During Field Studies O'COOOOOIOOOOOOO0.0.0.0000... Effect of Experimental Inoculations of Whole Tomato Fruits .0.0000000000000000....OOOOOOOOOOOOOOOOOOO. Mold Count in Relation to Percent Visible Rot in Whole Tomatoes Relationship Between Mold Counts and Percentage Rot of Whole Tomatoes Mold Counts and pH of Tomatoes Which Did Not Require Trimming and Tomatoes Which Required.Trimming .... Influence of Mold Genera on the pH of Tomatoes After Removal of all Visible Rot and Mold Growth Influence of Factory Operations on Mold Count and pH of Tomato Juice Flavor Changes Noted in Tomato Juice Prepared from Field Tomatoes Containing Known Mold Genera ....... PAGE 17 27 M7 51 Sh SH 57 S9 Organoleptic and pH Changes in Tomato Juice Inoculated with Molds pH of Tomato Juice Inoculated with Molds After Five Days Incubation at Room Temperature (Standing Culture) 00.00....OOCCOOOOOCOOOOCOOO0.00....0.0... Pectolytic and Cellulolytic Activity Produced by the Molds in Tomato Juice OOOOOOOOOOOOOOOOOOOOO..00... Polygalacturonase Concentration and pH of Partially and Wholly Decomposed Areas Trimmed from.Tomatoes... vi 61 62 79 101 PL ACKNOWLEDGMENTS The writer is very grateful to Dr. F. N. Fabian, dis- tsinguished Professor, Department of Microbiology and Public £€ealth, who suggested this investigation and who during its czourse made many valuable criticisms. Sincere appreciation fiLs extended also to menters of the Tomato Research Foundation caf Elwood, Indiana, who by financial support made this study goossible. Likewise, thanks are given to Edwin C. Fettig, ESecretary of the Tomato Research Foundation for making avail- eable the laboratory in his factory in which the field work fives done, and to Virgil Ray, Chairman of the Tomato Research FRNJndation, for his helpful guidance and untiring interest dixring the course of the work. Acknowledgment is made of the substantial assistance giqven by Dr. E. S. Beneke of the Department of Botany and Pliant PatholOgy, Michigan State University and his former EIWaduate student, Dr. Seth hizuba. Both participated in the f1-<‘31d and factory Operations and later identified the molds iSOIlated. Dr. Beneke kindly supplied the Kodachrome prints. Rohm and Haas Company, Philadelphia, manufacturers of mlzynm preparations and Sunkist Growers, Ontario, California, nmrnifacturers of pectic substances were very generous in sup— Plying experimental lots of their products and information CO‘i'lczerning their characteristics. vi INTRODUCTION It is well known that some molds have caused extensive damage to foods. It is not so well known that other molds have imparted very desirable characteristics to some foods and have resulted in the attainment of qualities which other- wise would have been impossible in these foods. The familiar Roquefort, Camembert and Blue cheeses are only a few examples of food products created by the activities of different genera, species and varieties of molds. Molds also have fermented foods to yield beverages, some of which have been consumed ex- tensively such as Sake, the national drink of Japan. In addition molds have been used in the manufacture of gluconic acid, citric acid, fumaric and gallic acids which have been employed in food products. By-products of mold metabolism.have been used on a large scale in the clarification and stabilization of fruit juices, jwms and Jellies. One of the characteristics of molds is that usually they grow more slowly than bacteria and yeasts. Some workers have used the presence of molds in processed food products as an indication of the condition of the raw produce and the efficiency of trimming and sorting procedures. This criterion of quality was applied to tomatoes and tomato products over #0 years ago and still is employed by government agencies. In recent years technologists have made considerable progress in developing improved tests for measuring food quality. Many tests formerly employed have been discarded as a better understanding has been gained of the basic prin- ciples of food production and manufacture. This work was undertaken to study the types of molds found in tomatoes, to determine the ability of these molds to cause enzymatic and other changes in tomato fruit and to relate, if possible, the findings to currently accepted in- terpretations of the Howard mold counts. The tomatoes were collected principally in the State of Indiana during the 1953 canning season. In terms of dollar value, Indiana is second only to California in the production of processed tomato products in the United States. Processing of tomato products is a major industry as is reflected in the value of the tomato pack which rose steadily from 19 million dollars in 1935 to 13h million dollars in 1951. PART I STRUCTURAL CHANGES ASSOCIATED WITH THE PRESENCE OF MOLDS IN TOMATO FRUITS Introduction The purpOse of this investigation was to examine the types of molds which are commonly found in tomato fruits and to study what structural changes may occur in the tomatoes when mold growth is present. The presence of mold necessi- tates extensive sorting and trimming in tomato processing factories as all mold filaments, regardless of type, are in- cluded in the Howard mold count. This has been used to con- demn tomato products as unfit for human consumption. Review of Literature Most publications concerning molds common to tomatoes have been concerned with the individual genera and species of molds which have been known to cause disease or decompo- sition of tomatoes. Some of these publications have been written by plant pathologists or workers who have summarized dat£1.for laboratory personnel engaged in mold counting pro- cedures. The technologist who seeks information concerning the effect of molds on tomato fruits usually must ponder through a maze of data which relates also to leaves, stems, seedlings and.roots. Most of these publications describe also the physiological, viral and bacterial disorders of the entire tomato plant. Unfortunately also, workers frequently have failed to relate the scientific name of the mold found to the disease of the tomato, therefore, a disease has been known by more than one popular name. Doolittle (19h8) compiled what has been perhaps the most ‘widely referred to list of phyto—pathogens common to tomatoes grown in the United States. Berkely and Richardson (l9hh) issued a similar summary of fungus diseases common to tomatoes in Canada. Workers at experimental stations have considered princi- pally the molds on tomatoes which have been responsible for financial losses in individual states. The publications of Young (l9h6) and Young g§_§1 (19hO) and of Davis (l9h8a, b, c, d, e, 1952) are pertinent to this investigation. MacGillivray 22.3; (1950) described symptoms on tomatoes affected by Phyto- phthora infestans, Alternaria sp., Pleospora sp., Rhizopus sp. 8nd_§h1tophthora capsici. Beattie gt 2; (19h2) listed only Corletotrichum.sp. and Alternaria solani as fungi attacking tomato fruits. Linn and Wright (1951) presented valuable data concerning several tomato diseases. Some investigators have published information concerning only one or a few of the fungus diseases observed or a parti- cular aspect of a tomato disease. For example, Henderson (1918) listed some of the characteristics of early blight and Phytophthora fruit rot found in Colorado and Heuberger (l9h9) elucidated the mold problem in Delaware. Middleton and Kendrick (1953) discussed what has been accomplished on tomato disease in California. Cunningham.and Lambeth (1951) gave emphasis to tomato disease control. B. J. Howard (1911) in the Food and Drug Administration of the United States Department of Agriculture introduced a laboratory procedure for counting the number of mold filaments in tomato products. Subsequently the Governments of the United States and of several other countries seemingly have come to regard the Howard mold count as an indication of tomato qual- ity. HOward (1937) gave some of the characteristics of several genera of molds, notably Alternaria sp., Colletotrichum sp., Fusarium sp.,‘Mgggg sp., Rhizopus sp., Oidium.sp., Penicillium sp., Aspergillus sp., and Botrytis sp. He pointed out that Alternaria, Colletotrichum.and Fusarium cause most of the injury to the fruit before the tomatoes leave the field and these molds seldom develop markedly on fruit which have been taken from the field in sound condition. Eisenberg (1952b) was another worker concerned with the Howard mold count and its application. He listed four molds which commonly attack tomatoes as Alternaria, Colletotrichum, Phytophthqgg, and Oos ora, and described the area affected and the depth of visible rot produced by these molds. A publication by the American Can Company (1950) reported characteristics observed in the tomato fruit when species of Alternaria, Aspergillus, Colletotrichum, Fusarium,quggg, Rhizopus, OidiumI and Penicillium were present. It was mentioned also that infection by Alternaria could be con- trolled by using resistant varieties of tomatoes. In a similar publication Troy (1952) of the Continental Can Company included the following fungi: Alternaria, 00113: totrichum, Fusarium, Mucor, Rhizo us, Phytophthora, Stemphylium and Cladosporium, and gave some of the effects of these molds on tomato fruit. A limited amount of information has been available in the booklet, Micro-analy§i§_g£|Eggd‘ggg_Drug Products, issued by the United States Department of Agriculture (1951). A summary of the most pertinent information noted in the literature is presented in Table I. .padmos moefiuosom mooapoouaa masocooou use pagan one ouca hadooo opennesod no: mooo maaaocaoao nowadm .nuOQm on» no command on» :o ooosooad mopedm .aoaoo aw sooam Cameos muons oasoad doomed» .muwdnu moanedan CH .mpasau Hausa Ho Odds» on» so moanaAdHomonaH ensue Guano hemp .oonoaaoo spoon muoaosdc can: .ndsaoo name one no Moon madame» one wcahno hp ooze Inmsoa uoSooon commune one .GZOan Seahehm osooon one season maepwnauoo oaoa maopnoo o>so «poem aooao .Goxcdm haunmaan one spoon Ho amazon .aOaGoo ends: on» Ga none none oeonaau: madam e couuo .maoucoo adamant one whooaon .oxo oudfiop moods Qua: nepoedao :N\Huoa\a oEooon Gwen one odd: he Eduaoquoaodz as emcee» honed .nxoodm can on meam .3oaadnn osoadm moaOQm szosx haaosaom .Haaam .QOpaH doao>oo muoau use oomoad used has no .oweHOpn hope: ox con: mSOpdSho on zone has nausea magma .owepm has pd .pamasap .uoHOHm oudfiou saaenaoaa< hand Gd .uooao one so one no» names: one .sfisa pop madam .ewcaxhqa can» on» no haaesns .ocHI mn,osoadn poms nomads unoocoo and: ooxaafi npzoaw hue>ao> nmaszoan .809» one on aces nosedm .Haou on» unmaan haaem a me wcaAdOQQe noose oouoouud one nho>oo unsaved no adaom ca manna» nomad Andean: maoaeadaoo Congo nomadm .pH:AH on» no on» as axosuu< ooheooo no dead .b one .HHW~ macaw on» can“ npmoo oSoa oa monopno heoeo .xoeao so ocdoz :do Enasdmao as on soaao a who mama .pasau on» no done M\H o>Ho>dH Haas» .ocdou .usoawoa logos: as czonx he: .osnnau pasaw aouoo on» haso poeuh< as nausea coup waazoam OpesOp haaoeaom .Anapae: .haonaeoa .uuoan adasoaao .xoean Condom noeucH esope woos a“ unopxo .G was .HHm~ .nos«> on» Bonn maoasaeanm mono has paohm nwdoaap uneven 050» on nasooo asdHOm «wae2h09H4 ooxoeppd pagan . oneouao no comm awwnm on» so asepmmum no hmOHOHEeofidm Ho oEdz coESoo emeum one Enacswao mmmmmoz mDOHm<> Mm nmeoz m4 BZMmHmm mH mazomc GAGE zam3 mEHDmh OBo noeuna nnHeEon mHSneoddm .nooau on» opnn hadooo mopenponod maunonvonu enwnnm .pa mnHonouKo hapeonw nonpoouna one nanno>o nee ednopoen one Hwnnu hneonooom .omnn own on ad and» on» he non hes pannn onapno on» one nopmoe hen» can» nn wanna nnonp noeen muons one nonz .neone oomeoe none: nomnea oeeeo one ooeoaeoo spoon one .oudeaaoo annnm on» no oedema» on» ownedno muons u< .oeoeoond one newnen on» no eonOQu noes: nn nodoon one Sunni mmoodu uneo no nonenn e zone one neg onooon eoSapoSom enepnoo .mwnaxnes wnnn cananoonoo o>en one oeeeondoo oaooon noon anode omens .noueneao :H on M\H no cane eueEapHn noeon one pannu edan aneon no edHn no haonden owneano euodm .pflnnn damnaa e no spoon onon no Ono .nopeseao na =m\J .nn«Soan noxnne mapnwaae .pnoonannenp .edodoadenoona .neanonno .aHeSn I Hmmmm .nonpeoz mneo .Snez .oopsoon anode on hapnoneonn HAM: unmanno>o awed eoOpeEop oopnon .odnnuoom .unoap uaonoo hnenaono noon: nunnm noonw on» ooe>na non nooo Seanemno on» hapnonedd< .ednn maneon no edan one unfinnh .msnonn eons" unann haunonedde eouenuoned unwnnm .unOan wanna one Hanna zone non oo npOQo pen ouannu noenm soups. an: n« we enoapaonoo oaneno>eu noonn owden unenaoao>on .Hnom on» nonn oEoo on uneomde nonvoounH .nwen ma nuannn nonuo cu ooneeadn one anode on» Eonh nonoam .onnnon oueEou no one» unanno>o .Haon oonneno thOOQ no nnooo on maoxuq .eeuepm Henpnoo one oneneape nH edono wanneo on eouooa neonnou ooeneo nopno .oopenanpnao mason: .nosaoo ouonoennpne nopnon nooaofion sesonopoponnoo nannm on» no“ “ale. 0. Ram. eoaomos< pagan no eweum hmOHOHSooadm omeoean no poem no onez nonaoo one nuanewno A.»noov H mumonn oomeoeao ooaxnanz.hapnwnao e men doom .npOdo .ono nope neon o>nuonn9moo ooxeom none: snnnn .noonw keno a Hanem epnepm naaenen noon on» no one onoonopo semnno open hnemon h.pnoz~ onepoo « onospsoo» no neon haaenen non enonm onmnnn on» no nonone nonnoz nonuo ozown wnanaeanoo moanaan Medan enononnn no oxoeno noun e>aponnpooo neon has nuns: epone noxnnn noeam nwnonnu onopnm efionm pannn on» ounn omen hen anon eunean oopoonne on» nn hHOpH nonnem naono>on nH .Sopm unaneona pooEHe one noohmm one swoonnp osnn coo .nooo Anonunomoown .m~ an uuoon one now we onapoe ennno o No ooupOQn non ouunnm nmnonnu unopnm anon unwnnn Enaneunm oomoepp< pfinnm ooeooaa no ponm pannm.onu no mammmmuml. no thHOHnoondm no onez nonsoo amuse one Ennnewno H (Hi A.pnoov H fldmda lll‘I )IH' .u nnn ooppon on no noaoneo hen nuzonm on non opnns nonmenm one nonpeoz pox nH .neonde enoapenoe epannneo .hazoae unoaoneo non non: .npaoo canenooaonoo e on pannn on» opoonne haaenen .1 one honopnnenoo na anonpeoa haaenen en pom 11 .mnawnen one we noxnne hapnwnae one one ooennne oononmnon e o>en muons coon: non pnwnan oped on peenpnoo n« enamnen oonnnoo nannene pon pen npoone e men annnn an none on» no ooennnm .emnamnen neafinde zone maaenoneeooo onepoonnn enonwnnophnm new: oouoonna npnnnn .nonozom ounnnnoo on o>en he: .uannn on» no once no naen no>oo he: omneano eponm noonm nonnenm no Adena e on Soudnne penan .noaaoonna nopne onnp oEoe non anon onoo Ion pon oo opnnnn noono .mnaxoad 9e euoo nzonn oappaa no anounnmo zone hex I NflMflW .Snan nnenon anonpnon oopuom .oopoonne on unnnn on» no aae no none .nnpds ooeondm .onoo m snnsn: snonn e no N\HIM\A no>o oeonno he: I nopeq .pon onoxonn oonnwnann openao .onen on» ooeonao on» onnw pen» eonen Inon neO Seanemno openou noxneo zone mHHenen pnn ownnxneso.oopoonne on men .nonpeon Enea na haoanen noono .nonp ouno Jone ooxeoe none: nzonn no oonoeade 30o oeen .ono Eoeooan on» we unnnn on» no ooennne .Haoe oonaeno nanoon nn one nonueoz no: .Ene: no noonnon oownoaonn on poonnne mnonw Ion nu unoaebonn poo: .Hnon on» n« eo>na nnmnnn .owe Inouo na .paenenp on .ononn on» on nuannn onan no pen» ooonp no Anon on» none» pen» eufinnn mmoeuue onwnnm .nopez hp oonOQm .nnen one onnz he oeondm .oneenp guano oooo an onnnnne ou_Eooe non moon .nOHp Ioonnfi nopne nee: e none“: heooo A.nnonm enumeonnou .no .aoon eoapnnenmm onospsdoonsm unnnn on» no nsounnwml ooxoeppe unnnn no oweam hmoaoaaeondm oeeoeaa no poem no onez nonEoo one Beanewno Aounoov H Ban. 2 1 .oaon Hennopxo anonpaz omen nope: Opnn oowneno on men pannn on» no moneoo menonsno.soono son: ”swoon on» no oooeen ownea neon nouno epnnnn mnnppom .pnnnn onnpno on» monpmoo onnon son e nH .ueone oxHHInopoaHn ee pnepm I Nanem .monOQm one oaon opnnz no nohea ooennno e zone eoEapoEoe a«0e no: mnnnonOp nunnnm .exoeno on» nH doao>oo hen eauonoaoo nun: oaon nZonm .pon ohoxonn Bonn ooeoeao neanwnnpono mmnnxnen nonnenm one nozonnen one ooennno one no wnannpdnm .nodo meonn haunonoonn enounoo one nSOnn mneo enooon on one» mmnnmnen one noponeao na ea onooon men one maaenoenm owneano macaw I nopeq .pon ohoxonn no omonp nenu nonaoMOp noooao one nozonnen .mmnaxnes openoe oonnapno mannenm onen macaw .oneno neHnmonnH no neanonao one: .nouoneao nn eHI:\H onooon eeone oopuom .omnan oanpnoonoo nzonn xneo one nzonn unwed wnapennopae onen eponm .nzonn o H ne oooenp mano no enounnno on zone he: I H m eonnoz nmnonnp oHoE oeonn noEEoo on» on Emanewno one .omenopo .pan Icon» .ononn one nuannn onnpen nH eoopeSOp onan haneon unopnm .uannn ouno ooneeane anon ononz no anon nun: poepnoo nn pannn no ooennno no ennooo .oonnoz nwnonnp no one Inooado nononn In: nmsonsp openponon no: .pnsnn onnn no noono no wnnnonfin.no .Hnoo oonneno manoon .3oa no one po3 onneEon HHooInou nan: nonpeoz mnnen nH-noEEoo poo: .oaonn on» nd annnw one ooouenou nonononz non nn pnoeonn on on ha onna on Emanemno .omenOpo one .onoeonp .ononn on non unmoummm .n» on ouunm non eunOpoouunm eon nnom n Mneamo ennopoosnnm ensue one do gsooonwmwl ooxoeppo pannn no omepm n.9noov H mumond poo: .onnn IeHoE uneonnne eonwnvom .noeo zonoo genome onoenn none: .nonnennpano Haoo one nnen he oeonnm .oSHpoEoe non anon on» n« onna nee noun: enuonOHoe no oeonne onH< .neon on neon Sonn noon: neone oopoonnH .Haoe nmnonnu oeonnm .eonone on hapno Inenn< .enOfimon wnn Izonw oueEou nonuo nn onen nHonupenen unwnan nnonunom Inoo.eouepm oopnnb .ooee HHoo nonop nnonunoe nn unOpNo «mnnmon nannn onen: oEoe on ennooo Enaponofiom ooaooope ononn annnn on» no enounnwmlix no hmoaownoownm omeum oeeoean no poem no onen nonEoo one Sennemno A.unoov H mdm N Z < Cit-302500 ‘U 0 2 C, (In: a» 5 splits, black 1" soft spot, dark center 1.1/2" black spot - 3/h" dry, black brown sunken spot cracks at top 1” dry, round, sunken spot black cracks at stem end cracks at stem end 3 wide splits in ripe firm tomato, covered with mold 1/3 sun scald, covered with fungus growth 3 cracks, 1" diameter 2 black, hard, sunken spots, 1" diameter sunscald on 2-1/2" diameter with black markings 1-l/h" diameter sun scald area with black markings black sunken area on 2 x 1/2" sun scald area 2" sun scald area, with black growth in this A splits, 1/2 x l" with Alternaria growth cracks with black mold in these growth cracks with 1/2" black spot in middle of sound fruit 3 splits with Alternaria, soft spots 1 diameter around splits 1-I/2" x 2" sun scald with Alternaria on surface 2" rot, hard on surface, also split 3/8 x 3/8 sunscald with Alternaria dark brown to black, faint concentric rings in sunken soft area 1/2" diameter 5 tan brown spots about 3/8" diameter 1/3 soft, 12 spots 1/3" diameter, sunken light and dark concentric rings 6 spots, sunken, light and dark rings darker in center spot 18 TABLE II (cont.) OrganISm Isolated Cello totrichum E ZZhNnHon U) :UD'UO p oozsz nH m ammo am > Tomato Appearance of the Tomato ___ h spots, sunken, firm, black smooth surface 2 sunken spots, dark and light rings 1/2 rotted, dried black surface 3-1/2” brown spots 15 spots l/u to 1/2" diameter 3/h" black, sunken spot 3/h" soft spot firm, brown area, no surface mold 1/2" spot on ripe, firm fruit 1/3" sun scald, 2-1/2" diameter with 3 small 3/8" sunken spots on the sun area l/h" sunken spot, 6 in number 15 spots, h of these large 1" diameter 9 spots, 3/8 x 1" diameter (Stokes) 15-20 spots fused together to make up 2" diameter Spot (Stokes) 1/2 soft rot 1/3 soft rot, insect injury, white hyphae on surface nearly all soft rot, pink white hyphae 1/2 soft rot, in two large holes, white-pink hyphae in holes 1/2" hold with 1” soft area 1/2" soft spot with firm skin 1/2 soft rot 2" in diameter 1" rotted spot, black, pink-brown surface mold cracks at stem end, pinkish-orange mold, some shrivelling cracks at stem end 1" round, soft spot covered with .grayish yellow mold 1-1/2 soft spot blacktcracks at stem.end 2/3 soft rot, gray brown mold 1" soft rot 1/2" deep, insect injury - soft rot 1/2dsoft rot at blossom end, with deep crack 1/2" deep, insect injury, soft spot 19 TABLE II (Cont.) Organi sm Isolated Tomato Appearance of the Tomato Fusariinn deep, l/2" insect injury - soft rot 1/2 soft rot at blossom end with deep crack 1/2” deep. insect injury, soft spot 2 holes near stem, soft spot 1 1/2“ very soft with 5 cracks somewhat soft with 3/Lp‘ dried shrivelled spots 2" soft rot area on side near split 1-1/2" area soft rot 1/2 soft, 1/2 rotted 2" split on side, 3/8" wide 1/3 soft rot, l-3/h" diameter‘ soft, dark red spot 1" diameter 3/h" soft rot, dark red color Mycelia 3-1/h" spots, light brown éiprijlia 2" soft rot area with h spots in the middle (Stokes) ~_—------—--—--—--- l-<>< ¢<£so£ spas .Esao . =N\HIH .so some venousoooo coxndm some swam :d\H I 0 mm = a omcmno 02 I 4 = = can =0H\H I m npzonw .Hm hao> .dEoooo :HIN\J .wo owcdno 02 I o m: = a z = z mwfiwflo 02 I < pace Sea: a = a z = noao>oo some =:\H I m .oodd no pGAOQ as spzoam m>mon oHoS and: and: oomOQEoooo some :N\HIH .Noadd4 oeno>oo some =:\A I 0 mm usHHthommfl pom oodmade groan anon - =N\4Ia adonw .moam flamenco made =:\H «one soH\H I 4 33 same use: .sfie gm} omcano oz I m dons undo usmaa Isaac :M\H some domes» =:\H I 0 mm swam omas . odou ooampso so“: some Medan peak sN\H «can :oa\a I 4 . econ asapcoo madam Spa: some psau =N\H omnsno 02 I m mead :oa\a I o «m econ pmau an ooocdoaASe .Sdao :N\4 Madam .Esaaoohe HasmH> oz owcano oz I 4 Isaac =:\H Medan .mmaw nomads oz .Edfiu =:\A I m .Esao =:\H madam .soas nomads oz smudge oz I 0 an «Hascuopa< «use 0 ad accessodd< ammo m as coasasodd< condom Emacdmao mBHDmm 084206 mqomz mo mZOHadqboozH Aonm o as mean «one nzonn onwaa .oonoppaau =m\m ha coondonnSm mono xnuo :m\4 .oona no opam pm pnaod nag pdooxo eopoonnu sane :mm\a I 4 omnmno oz I m wnanoxowan oz .oopooumm sons ::\H nouoouus sons =Nm\d I u m: Ennoanpopeaaoo ownmno on .Eaam omnmno oz I < oomodSoooo one muopu3 «one wean =:\H I m comednoooo one znoumx mono umam :m\a 0 mm humus: one oomoanooon npzonw nu“; dons =N\H < muons: one oomodaooon Anuzoaw new: «one :m\H m muons: one comognoooa npzonm nun: sons :m\H 0 on omnmno on .Enaz omnmno oz n oomoasoooo one human: mono paau :w\a m mamas: pon unn .aHom omndno oz 0 m: mapnhpom when m as eonsnaoddd muse N as oonanwoddn condom .nmanawno o A.»noov HHH mqmeom pad =:\H I < noxnne non .eoue . mneo :N\HIH ho ooonnonnne eone :m\H pad :m\4 I m oaos cans: sea: oonoooo one 2 \H pao =oa\a I 0 mad npzonw oomOQSoooo penSoEoe one unopez oaon m>eon pen .afid oz I < eene oeeOQEooeo .xneo :N\HIH hp oeonnonnne _ .oocw no pesos om nozonw pesos can oao =oa\a I m onenzomao awash mo ooh op peenunoo nH .eone and unmad :N\H an ooonsonnne I .oona no ocaoo om sozonm ocaoo can one =~m\a I 0 mm oocoooeau so: .eone soean =:\H one =mm\a .oaoe I e znouez one oomodaooeo zaonapnm wnappad on .oHoE I m mnepez one oomodSooeo zaonapnm wnappad on .oHoE I o 4N Enaneesm odds opeEop no peoz .onou neonm ®\H mp ooonsonnsm .oonH mo unaom ue eone =®\H xoeam owneno oz I < ssnoanpopoaaoo eheo m we ooneneodd< ezeo N pe ooneneedd< condom Seunemno A.unoov HHH mqmeon zno> oouenwnepnoo oemoeno .znouez ooeaen z>eo£ zno> oexoeno .znouez oomaen z>eon zne> ooxoeno .znouez oeeaen z>eon ano> oepenasepnoo .ua wnano>oo manned .oneEou no>o Ampanzv Epsonu nen npzoao nee nuzonm oaan pannm mo ooeMQSm no>o .nzooxeonn enca>oo oz .oona mo pnaOQ pe npzonm z>eon zno> ooemnnm ne>o .nZOoxeonn enca>no oz .oona Ho pnaOQ we anOnm m>eon zno> pmox .eone nmazoaaem :0H\H zn ooonSOnnnm eone nooaw :oH\H npzonm oHoE opfinz .meoaooonne oconoonao an oaaom sauna npzonw oHoE open: .meoaooonao ozononoao an peso» oases wnwn peau .xneo eN\H moan pane .anee =m\a mean omen .anee =m\a mGHH peak «Shed :N\H mnan peak .xneozN\a wnan peau .zneo =N\H oaos onom .eone peau =N\H oaos once sot =:\H eone xneo ::\H eone xneo =d\H eone xneo =N\a mnfin xneo ::\H oHoows nH oHoE .wnan =N\H =N\H O£D<¢ m<: mod mNH zma UH ~.pnoov noon: ozeo m pe coneneodd< ezeo N we ooneneodd< condom Emanewno A.ocoov HHH mumee 31 eone nonpon n30nn ed up ooonnon Iadm .neanmonna .npzonw oomaen ::\A eene nonpon nzonn ea mo ooonnon Inna .neanmonaa .npzonw oomaen ::\H eone nonpon ea zo ooonnoansm .nuZOAm mean: :oH\H .oona mo pnAOQ pe eene noenm =o\H pdooxo emneno meoa>no oz oooneonnnm npzonm open: oomaen oepenanepnoo nascnm msoa>no on .pon oz eone peam n20nn =:\m ha ”MS eone noanm .nzonn =N ooonnonnnm npzonw mean: nmanoonw =N\a eone nzoan .ooaxnanz sN an ooonnon Inna .npsonw opanx noonw .zenw ::\H eene nxonn .xneo :N\HIH an oeonnonnnm eene mean: :Nn\a canaea> eaaoozn no manna oz eone nzonn :N\H mp oeonnonane eone dean: :Nm\H owmhd nacho ::\aIH zn_ooondonnse eone :Nm\a eone peak .nZOnn :d\m mp oooneonnsm nuzonw mean: oceaen =m\H nozonm ooanz smaeoonm =m\a eone nzonn .ooaxnanz 2N zn ooonson Inde nuzonw moan: noonm .zenm ::\H and aoH\H one =oa\a one =e\n oao :m\a omneno oz omneno oz one :mm\a one goa\a one =Nm\a owneno oz emneno oz eone =Nm\d owneno oz eene peau :N\a eone wean ::\H ezeo m ue ooneneedd< ezeo N pe eoneneodd< A.oooo. HHH memes e m o omH < m 0 «ma eanouoouanm 4 m o HHH < m o mN < m o Bea Edaaaaodnom oonnom Sednewno II|1 32 nepeaeao :m\4 .eene xoeam eene mneo :m\4 4 nepeneao H.eone xoeam owneno oz m nepeeeao srw .eene amen omneno oz 0 mm eEneoonoHna npzonw oeeOQSooeo one knead: nose .eene aneo ea 4 oeeOQEooeo one znouez eene xneo ea m oeeodaooeo one znepez eone xneo :N o Hoa npzonm oeeoaSooeo one znepez enom eene xneo :H o npzonm oeeodsoooo one hampez once eene aneo :N m oeeanoooo one undue: eone xneo 2H 0 mm eene oeeOQEooeo one znopex oenoaooeao .ueam :H 4 eene oeeodSooeo one uneven oenoaoeefio .ueau :N\Hva m oee0deooeo one anepez noauenoaooeao ea o How unmouanm nopeoo oeanz =oa\a eon: eone some =m\a :oaoenoaooeae =e\a e owneno oz owneno oz m eone anon :N\._” 55:03 zn oeonnonnne npzonm ouanz =m\4 emneno oz 0 do ednouoouanm emeo m we ooneneodd< ezeo N ue coneneedd< condom newnewno “.oeoov HHH memes 3.3 nepeseao :m\4 .eo eene nxonn .weona ho oeonnoaane .eone mneo ::\H oen awash mo amen .eene noonm =d\H zn oeonnoanne .eone xneo ::\H I Ii.l'...i!\9¢l‘fl .1... 1.!l I.‘;l‘:!t '1‘ il‘ eene xneo :oH\H I 4 eene xneo :oH\H I m oen pannn ho ween .eene neenm “.onmbw =:\H mo oeonsoaane .eene xneo ::\H owneno oz I o H: efineoonofina ezeo o pe eoneneeddn ezeo N we eoneneeddd condom sadnemno A.pnoov HHH mqmde Aspergillus Bot is Fusarium Mucor Fig. 3. Effect of experimental inoculation of tomatoes with Aspergillus, Bot is, Fusarium, Mucor, Oos rs, Rhizoctonia, an Rhizopus after days at room temperature). .Ooeggra Rhizoctonia Rhizopus -Fig. 3. (Continued) Effect of experimental inoculation of tomatoes with As er illus, Bot5%tis, Fusarium, Mucor, Oospora, Rhizoctonia and hizopus a ter E days at room temperatures. r— Alternaria ‘ Colletotrichum Hormodendrum Fig. h. Effect of experimental inoculation of tomatoes with Alternaria, Colletotrichum, Hormodendrum, Penicillium and Trichoderma (after H days at room temperature . 37 Penicillium Trichoderma Fig. h. (Continued) Effect of experimental inoculation of tomatoes with Alternaria, Colletotrichum, Hormodendrum, Penicillium and Trichoderma (after H days at room temperature). I. LIkai. i lh-....Ylhl.J ., I .I II. 34%,)! x f . . It.“ ‘FIW. L831”. . 38 Discussion This study has shown that considerable training in mycology is necessary to identify the types of molds which may be present on tomatoes. Cultural and microscopical examinations frequently are necessary for positive identification of these molds. The genus, species and strain determines whether appreciable dam- age is done to the tomato fruit concerned. In addition, the variation in the severity of attack would be influenced by many factors, such as the variety of the tomato, soil and weather conditions existing in certain areas and spray programs. The rapidity of handling the tomatoes after they reach the factory is also very important. Excessive humidity promotes mold growth (Block, 1953). Yet, unless the relative humidity is high, tomato fruits will have excessive loss of moisture to the surrounding air and in time most of them will become wilted and shrivelled. Rose 23 2;. (19h9) reported that ripe tomatoes at 60°F evolve approximately souo BTU per ton per 2h hours when stored. The Howard mold count makes no allowance for the signi- ficance of the types of molds present and, in any event, the average mold counter would not have the experience to identify the types likely to be encountered. It is obvious, however, that it would not be reasonable to regard all molds as being of equal importance in causing rot in tomatoes. It has been 39 observed that molds in the fields and in the factory produced characteristic changes in tomato fruits, some severe, some mino r . It was interesting to observe that some strains of Rhizogus produced cracks in tomatoes. It appears that even a minute perforation of the tomato cell wall which allows the introduction of this organism may be the precursor of exten- sive damage. No reference to this action has been noted in the literature. Instead, it has been mentioned that molds grow in cracks; and that the cause of cracks in tomatoes has been obscure. It was suggested that soil and weather condi- tions may be involved (Howard, 1937). It was interesting to note also that under experimental conditions while many of the molds produced irregular lesions in tomatoes, strains of Aspergillus, Colletotrichum, Oospora and §_e_nicillium appeared to produce a metabolite which was radiated evenly throughout the tissue to give circular lesions. It can be appreciated that considerable difficulty would be eXperienced by a canner when many of his incoming tomatoes showed Colletotrichum phomoides spots. The number of these blemishes on a single tomato were shown to be numerous on occasion, and would involve extensive trimming which would substantially increase costs of production. Another consid- eration is that in the early stages of attack by this organism relatively little damage appeared obvious in the tomatoes and LIO might be overlooked by housewives commonly employed in trim- ming and sorting in the factory. The appearance of these spots on the canners stock would depend on soil and weather conditions in his particular area and might be sufficient to force him out of business when he is in the position of com- peting with processors in other areas relatively free from the effects of this organism. Other organisms may present a similar problem. For example, Alternaria often was apparent only as a small brown or black area not unlike a scar at the stem end of the tomatoes. It would not appear reasonable to always interpret the mold filaments present as an indica- tion of a rotten condition in the tomatoes. It was interesting to note that similar blemishes were observed on prize tomatoes on exhibition at a state agricultural fair. There appear to be many conditions which may influence the development of molds. Brown (1922) showed that certain volatile substances given off from plant tissues, especially when the latter are bruised have a distinct effect on the germination of fungal sPores. Distinct stimulation to Botrytis was provided by apples but depressed by potatoes. This stimulative effect was more readily discernible when the spores were of feeble ger- minative capacity. LII Later Brown (l9h8) reported that if a host is held at warm temperatures, resistance could yield to susceptibility and that pathogenicity of fungi may be greatly influenced by Varied illumination, manuring or even the presence of another Parasite in the tissue. He emphasized that "stalling sub- stances" produced by a fungus may inhibit its growth. 14.2 PART II MOLD COUNTS, FLAVOR AND pH CHANGES IN RELATION TO STRAIN OF MOLD AND PERCENTAGE ROT PRESENT IN TOMATOES 1&3 Introduction Following a packing season many canners have been con- fronted with Government seizures of their processed tomato pznodhacts. Frequently the basis for these seizures has been that the Howard mold count of certain packs has exceeded tolerances established by the Food and Drug Administration. Many packers have claimed that it has been almost im» possible to meet these tolerances under certain conditions. Some processors have claimed that when a mold is present on fresh tomatoes, the fruit sometimes may have superior flavor mad.<3dor without any apparent loss in quality in other respects. YThis study was undertaken to show what relationship ex- ists between the percentage of fruit showing mold and Howard mold counts. It was thought desirable to investigate also, what ixfifluence, if any, the growth of various molds has on the acidity of tomato fruit and what changes in flavor and odor result when molds are present in tomatoes and in tomato Juice. It has been shown elsewhere in this study that molds may produce only minor blemishes on tomato fruits. Review of Literature Relationship between Mold Count and Percentage Visible Hot in Tomato Fruit After the passage of the first Pure Food Law in 1906, in response to numerous requests from industry as to how to test tomato products and interpret the results, the Howard mold count technique was described for the first time (Howard, 1911). In 1913, using the principles of the Howard mold count method, regulatory actions against camera were instituted but it was not until 1916, however, that the first mold count tolerance was announced. This was for comminuted tomato products and allowed 66 percent positive fields (Smith, 1952). In June, 1917, some of the more important points cov- ering sanitary control of tomato plants were stressed (Howard and Stephenson, 1917a). In October, 1917, again in response to many requests for definite details of the test, more information concern- ms the mold count method was given with a few minor changes (Howard and Stephenson, 1917b). It was stated that very few tests had been made to correlate the microscOpic counts with the amount of rot by weight when the Howard mold count had 15" n" .u n s .1. IA! IntTvO in VI n~.. . erg..." —.._ 16 been first introduced and data on this aspect was presented. Tests were conducted both on laboratory prepared and plant samples. Despite the fact it was somewhat difficult to pre- pare in the laboratory a pulp of Just the same texture as that made under good factory conditions, the same general re- lationships between the percentages, and mold counts were noted. No laboratory samples with less than 5.5 percent rot gave a mold count of more than 50. No factory samples with less than about 14 percent rot gave a count of more than 60. In this same report it was claimed that no high mold counts occurred in samples low in amount of rot but that low counts were obtained in samples containing a substantial amount of rot. Reference was made to the fact that when stock is pro- perly handled the mold count is of greater importance than the counts of bacteria and yeasts in Judging the condition of the raw stock. Bacteria and yeast tolerances were given also in this report . Darling (1922) criticized the Howard mold count and as a result of many complaints concerning the technique con- ducted an investigation under the auspices of the National Research Council for the Society of American Bacteriologists. Be contended that the kind of fungus is important and that some hyphae break apart more readily in some instances than in others. Darling concluded that the Howard method for de- termining molds on tomato products could not be depended upon ’46 to give very accurate results. He referred also to sample variability, the type of fungi and the error introduced by different analysts. Later, Howard (1937) listed seven types of defective tomatoes most frequently described. The first Government standard for tomato juice was an- nounced on July 1, 1936. This allowed 35 percent positive mold count fields. Subsequently on July 27, 1938, it was reduced to 25 percent and on June 17, 19110 further reduced to 15 percent. On January 13, 1911.1, the tolerance was raised to 20 percent .(Smith, 1952). Eisenberg (1952a), Chief of the Microanalytical Branch of the Federal Food and Drug Administration, emphasized that low mold counts could be found even when substantial amounts of rotten tomatoes were employed. He mentioned particularly tomatoes infected with late blight (Mophthora infestans) but indicated that filaments of this mold in some respects do not meet the empirical rules laid down for guiding mold counters and that there is reason to believe that low counts from such tomatoes may be due to failure to recognize the f1laments of this mold. In another publication, Eisenberg (1952b) reported counts from samples prepared in the laboratory from tomatoes showing various molds. Some of his data is of interest and is pre- sented here (Table Iv). 1&7 TABLE IV MOLD COUNT IN RELATION TO PERCENT VISIBLE BOT IN WHOLE TOMATOES Taken from Eisenberg (1952b). Partial data only. ——f Organism Visible Rot Mold Count Alternaria 2.7 53 " 2.5 39 " 6.9 9h " 10.1 79 " 3.1 61 Colletotrichum 0.3 0 " 0.1; S " 7.0 92 " 0.6 18 " 5.1 143 ” s .9 57 " 3 .1 SS " 0.6 13 " 1.1 20 OOBBOI‘G 109 55 9.9 60 " 18.0 62 3 me 89 10.0 100 . I’ uu‘ r- '1. I b 14.8 Many articles concerning the application of the Howard method to tomato products have appeared in trade publications (Eisenberg, 1952a; Jones, 191m; Jones and Ferguson, l9h9; Jones and Pierce, 1947; Gould, 1952 and 19533 Siegel, 19514; Siege]. and Strasburger, 1951; Troy, 1950). These contribu- tions usually have stressed the desirability of keeping mold counts within the Government tolerances and have been criti- cal of canners whose packs have'exceeded these tolerances. Smith (l9h8a) listed Mucor, Aspergillus, Fenicillium, Oosgora, and Motrichum as examples of molds found in mold counts but did not describe the effect of these molds on tomatoes. Later he stressed sorting and trimming pro- cedures and presented data showing the distribution of mold filaments in tomato tissue (Smith, 191;.8 b). Influence of Mold Growth on Flavor, Color and Odor of Tomato Juice Culler 91 21. (19h9) believed that while mold counts have been adopted as a measure of quality, more should be known about the actual effects of these microorganisms on the products. These workers tested, in tomato Juice repre- sentative strains of Alternaria, Aspergillus, Colletotrichum, My Mucor, Penicillium, Rhizoctonia, Rhizopug and W in addition to other fungi. They examined the PH. color, taste, odor, and also changes in the refractive r-.. -a- a'se. ._v “ LJ b.1‘J‘w‘: -.' .. . w a..»' urai utq Is ‘I 1+9 indices - They reported that no off-odors or flavors were detected in the tomato Juice after incubation at room tem- perature for one week but that marked pH changes did occur. Kertesz and Loconti (19141;) investigating consistency said that little appears to have been known about the chem- istry and physiology of flavor in tomato juice except that it is influenced by the proportions of sugar and acid in the Juice. Experimental and Results Relationship between Mold Count of Tomatoes and Percentage Visible Rot Tomatoes showing the principle types of molds known to be present in Indiana and Ohio fruits as found in Part I of this study were selected from the factory platforms and from the fields. These tomatoes were red-ripe unless otherwise noted. The decomposed section in each tomato was cut out, weighed and expressed as a percentage of the weight of the individual whole fruit. The affected area was combined with “1° balance of the whole tomato and passed through a labora- tory cfirclone containing a 0.027 screen.* MOld counts were made employing the method advocated by the Association of Official Agricultural Chemists (1950). ‘ r «n- B Manufactured by the Cephaly Experimental Company, rentwood, Maryland. u 5 i ! s u I I ‘I a5 L I “e-ll a"’ ‘ a nu»- |;!' .404‘ ..o\ 50 Continuation of the type of mold present was made by cul- wring - The results are given in Table V. A Howard mold count of 100 was obtained when the per- centage of rot present in the sample was as low as 1.9 per- cent- (Table V). Counts in the range of 50-100 percent were noted frequently when the amount of rot present was less than 2 percent. This applied regardless of the genus of mold present. In one instance Alternaria when present initially to the extent of producing only 0.1 percent visible rot later gave a Howard count of 57. The high Howard mold counts obtained in this study when low concentrations of visible rot were. present are in contrast to those results presented by Howard and Stephenson (1917b), who noted that no laboratory sMples with less than 5.5 percent rot gave a mold count of more than 50 and that no high mold counts occurred in samples containing only a small amount of rot. The emphasis in published works (Eisenberg, 1952a; Smith, 1952) has been that a high percentage of visible rot may give low mold counts. This has been confirmed in the results obtained here. However, what has not been emphasized is that low concentrations of visible rot may give high mold counts. While Eisenberg (1952b) made no mention of it, his data showed that 2.7 percent rot produced by Alternaria gave a 51 TABLE V ROT OF WHOLE TOMATOES RELATIONSHIP BETWEEN MOLD COUNTS AND PERCENTAGE Mold Count Organism % Rot Mold Count % Rot Organism 868001 20 002820 no 860220000 022802 22020 98990026¢n~20mm 008970 60 680820000 0 11 1 11 1 1 1 1111 9683851595715 270289 30 861110858 eeeseeeeeeoe ease as eeeeeeee 5317255686305 Amos/731 I45 135673989 000000000000 11 13 1 11 m a 1 1 n 1 O m a 1 t 1 .1 r 1 C 0 runnnnnnnunl r0 an" Cflonnnnnnn runnnnnnunnn a O .1 z t m mm. m 1 m P HO P % C 0 069062200 086282897 0 08326008 1480608030 mm857m999mmgm28luu265 mmmm8288mm2%56mh22m6m 256053307h919916 17.4001 . C O O . O C C O O O O O C . O C O 7233 503215112h23021n3100 Alternari- a 698 98 972718u315870182 728201h§91h31 92 28.721 Colletotri chum 1 2 0':- 'I . iii-Ilia. aria . 1....th wxu 52 a count of 53, that 3.1 percent Colletotrichum rot yielded a count of 55, and that 1.9 percent rot produced by w gave a count of 55. ' It can be appreciated that it would be impossible for the average mold count analyst to differentiate these types of molds in a sample of a tomato product. However, it is the type of mold which is the determining factor in the amount and type of rot present, and likewise the type of mold has an important bearing on the mold count itself as considerable variation is known to exist in the size of the filaments (Beneke, 1950). The type of mold which the Howard mold count does not and cannot determine is a more important con- SideI‘a‘tion than the mere presence of infinitesimal mold frag- ments, which may or may not be important from the standpoint of rot . “019 Count of Trinmed Tomatoes Compared to Mold Count of TOMtoes Which Did Not Require Trimming It was thought of interest to compare the mold count of tomWOes which did not require trimming with those obtained by examining tomatoes which showed visible rot or mold which recmired trimming. Accordingly ten pound samples of tomatoes which showed many of the molds encountered elsewhere in this study Were collected at a canning factory platform. From the same hampers four ten pound samples which showed no visible HI 53 rot or mold growth were carefully selected. The lots of tomatoes which showed mold or rot were carefully trimmed. A11 sets of tomatoes were individually comminuted and Howard mold counts and pH values were obtained on the macerates. Also, to determine what influence, if any, the different genera might have on tomato sections remaining after the tomatoes had undergone trimming, decomposed areas were cut out from individual tomatoes and the molds therein identi- fied. The pH values and the mold counts obtained by com- minuting the individual rot and mold-free sections were com- pared. The mold counts of tomatoes which did not require trim- ming in all but one instance were lower than those obtained from the examination of tomatoes from which all mold growth ““9 rot had been removed (Table VI). These findings are in agreement with observations noted by Hand 2;; 2;. (1953). In this same study these investiga- “1'3 found that even after trimming, the mold count of U. S. O“ 2 tomatoes which showed 100 percent defects other than color rose over 20 percent six times out of 12 in 1950 and three times out of 12 in 1951. By contrast U. S. Mo. 1 and 2 free from defects other than color, without trimming did not Yield Juice showing substantial mold counts during the ”Per iment . S 5h TABLE VI MOLD COUNTS AND pH OF TOMATOES WHICH DID NOT REQUIRE TRIMMING AND TOMATOES WHICH REQUIRED TRIMMING —: E ‘— _;_ r ;- Tomatoes which did not Tomatoes which required trimming require trimming $2131. ( ioniidefifi‘égin.) pH 83161?“ (10811333231 te) pH 1 5 u. 3 1a 30 h-6 2 2+ h-B 2a Lu w. 3 8 m3 3a h hoe 1* 2 Li. 7 he 14.1). 14.5 W TABLE VI: INFLUENCE OF' MOLD GENERA ON THE pH 0F TOMATOES AFTER REMOVAL OF ALL VISIBLE ROT AND HOLD GROWTH N j genus isolated pH of rot and No. of rem discarded area mold free area tomatoes \ W (4.5 50 1402 LL -——'..c°11°t0trichum 14-6 12 m3 8 Wu 1P5 32 W “-5 “3 n “'02 2’4- 11.6 2 Ph “10 hora ’4-3 10 M; 8 ‘\ 55 It will be noted also (Table VII) that the pH value of the composite samples which required trimming were generally higher than those of tomatoes which did not require trimming. The majority of the trimmed tomatoes had a higher pH in the vicinity of #5. No individual genus produced a signifi- cantly higher pH than other genera. The high pH value in these trimmed tomatoes substantiated the findings obtained by the examination of the composite samples. Influence of Factory Operations on hold Count and pH of Tomato Juice A3 laboratory experimental results frequently differ from those obtained under practical Operating conditions, it was decided to investigate further the pH and mold count or COI'IIninuted tomatoes. At a canning factory ten pound samples were taken of tom‘i‘tbes as these arrived on the platform, after they had been “3th and flumed and after they had been steamed, culled and trimmed. Ten pound samples of the trims and culls were col- lectb‘d also. The samples were comminuted through the labora- tory pulper-finisher. Mold counts and PH values or the Juices obtained were compared with samples of tomato Juice °°118cted immediately preceeding the evaporator. 56 The experiment was repeated eight times. An effort was made to sample similar lots of tomatoes as these proceeded through the factory. The results are given in Table VIII. After the washing, trimming and culling operations there was invariably a reduction in the Howard mold count. A comparison of mold counts obtained by sampling at the other sites did not reveal any significant information un- doubtedly due to the important sampling error. The high pH values of the trims and culls was the most outstanding finding. All of the eight samples were appre- ciably more alkaline than samples of incoming tomatoes. The PH value ranged from (1.8 - 5.5 in contrast to the pH range of incoming tomatoes which was h.2 - h.3. These samples showed 100 percent Howard mold count fields. Under the fac- tory conditions noted trimming and culling of tomatoes made the tomato Juice more acid in addition to decreasing the mold count. It is suggested that molds may contribute to multi- Plication of flat-sour organisms on whole tomatoes in fields and in factories and thereby present a potential hazard to a Processor. Furthermore, if tomato Juice is made from poorly culled and trimmed tomatoes and as a result shows a high pH, then this Juice might bemore subject to spoilage by flat-sour 57 ooa m.m ooa m.m ooH m.m ooa m.: ooH m.: ooH o.: ooa m.s ooa m.m eases one mamas he m.: as m.: on H.: mm m.: a: m.: o: m.: mm m.: om m.: o: m.: as H.: .sassodsee oaouon ofidd OOHSH o m.: m m.: o m.: m m.: m «.3 NH m.: ea m.: m m.: assesses one .mda -Haoe .wss namepm neph< an m.: Hm m.e no m.: ea m.: as m.: as m.: om m.: as m.: wdessse one madame: sosc< «a n.: mo m.: mm ~.e s «.4 a: ~.s an m.: an m.: am m.s .onoeessd pcdoe canoe accoo assoc assoc added assoc assoc II. odes mo ones he uses no ease mo odes mo ends we odes no case we possess m o .m m a o m < ho condom v F 11'»?! ((1 aoHE. oases so so 9,2 .563 So: so mzogémmo amoeoss so 388st HHH> Mgm<fl 58 organisms some of which have been reported to spoil only tomato juice having high pH values (Pederson and Becker, 19119; white, 1951). Influence Of Mold Growth on Flavor and pH of Tomato Juice As a preliminary investigation of the influence of mold on the flavor of tomato Juice organoleptic examinations were made of thirty-five samples of Juice prepared from tomatoes which contained most genera and species of molds found in the Indiana and Ohio tomato fruits. The entire fruits, many of which showed advanced decompo- sition, were cosminuted. The results are given in Table II. Only 3 of 21 samples containing Alternaria sp. and Colletotrichum sp. produced off-flavored Juice. By contrast, four Of five samples of Juice containing 94352933 were bitter. The sample of Rhizoctonia which showed Off-flavor was nauseating. The sample of Penicillium showing “a“? Change was pleasant. S“haequently tomato Juice was prepared from sound whole t°mat°°a and dispensed in 100 m1 amounts into 200 ml bottles. Tm” were plugged with cotton and sterilized for 20 minutes at 15 p(Hinds pressure. Duplicate sets of bottles were inocu- lated with spores and vegetative fragments of 27 test organ- isms and then incubated at 20-22°C. 59 TABLE II FIsAVOR CHANGES NOTED IN TOMATO JUICE PREPARED FROM FIELD TOMATOES CONTAINING KNOWN MOLD GENERA 7* F1 NO 0 NO 0 NO 0 Orglni sm Pr 9 sent Sample s Samp 1e 3 Samp 1e 8 Examined Showing Showi ng Off Flavor No Flavor Change Alto rnaria 12 2 10 Colletotrichum 9 1 8 W b, 1 3 92922.22 5 u 1 Peni ci llium 2 1 improved 1 Rhizoc tonia 3 l 2 60 The inoculated Juice was examined and compared to the uninoculated controls at the end of 21.1, 118 and 96 hours. Four tasters participated in the examinations. The results are given in Table X. During the initial stages Of incubation some of the molds produced pleasant tastes but by the end of 96 hours bitter principles were liberated and many of the Juices had Changed markedly in flavor. Tomato Juice containing Penicillium showed a flavor which was preferred to that of uninoculated Juice. Rhizoc- 32915 SP. produced a very objectionable taste and odor. NO ill effects were noted after tasting of the juices. The tomato Juice had to be disturbed each time an organo- 1991710 examination was made and some bottles were consumed to ‘ 8’93ter degree and more subJect to agitation than others, Which undoubtedly influenced the growth of the molds and pH. It was considered desirable to investigate the influence of mGilda on tomato Juice when samples were left undisturbed: and to investigate further the influence of genus and species. The experiment was repeated employing 121; cultures. The Juice "as inoeulated with the cultures and incubated for five days b°f°r° readings were made. The results are given in Table XI. In standing culture Rhizoctonia sp. produced the highest pH value. Fusarium, Mucor, Oospora and Alternaria yielded pH vallies over 5.0 in most instances. TABLE X ORGANOEEPTIC AND pH CHANGES IN TOMATO JUICE INOCULATED WITH MOLDS 61 10 Organism 214 hours 148 hours 96 hours days [J3 \ W5 8p. - - flat 6.3 4.7 n - - - u.8 5.3 A8 "’ "' flat “.08 14.05 W SP. - - bittersweet 5.5 5.2 " niger bitter bittersweet bittersweet 14.7 2.9 clagifgrmus - sour sour, strong 14.8 .8 odor £01 lgtotrichuLn phomoides - - sweet 5.0 5.0 n - - sweet h.7 h.8 fix 1 - - sweet h.8 h.9 w W bitter bitter bitter h.8 14.9 , pungent odor " Just off bitter bitter . 5.0 pungent odor W sp. sweet sweet sweet odor 6.1 5.9 and taste 2139-35 89' sweet bittersweet sweet sour 14.6 14.5 u like cider sweet bittersweet sweet sour k.7 h.8 a like cider sweet bittersweet sweet sour u.6 5.2 like cider 9.5.2225 39- sweet sweet sour sweet sour 14.7 14.9 like sour like sour a milk milk sweet sweet sour sweet sour h.9 5.0 like sour like sour a milk milk h 8 h 8 - sweet sour sweet sour . . §g52£2§111£2gg spw sweet sweet sweet 5.2 h.9 a sweet sweet sweet fi.g fi.§ 2 sweet sweet sweet . . W \nigricans sour sweet sour sweet sour 14.3 14.5 n sour sour sour fi.9 5.9 3:; c sour sour sour .3 . W sp. sweet bittersweet bittersweet 1p? 14.7 with strong u odor sweet sour souri t h.7 6.8 unp easan W sp. sweet sour sour 5.3 14.9 unpleasant unpleasant 0 I; .1“. 2’1 III. .I.l .8! N1 62 TABLE XI pH 01'“ TOMATO JUICE INOCULATED WITH MOLDS AFTER FIVE DAYS INCUBATION AT ROOM TEMPERATURE (STANDING CULTURE) Mold “liar“ Min. Max. M sp. 2 5.2 5.7 M clavatus 1 5.2 W 5153; 2 3.3 3.11 l_8261‘gillus terreus 2 14.8 lbs W sp. 1 M7 Colletotrichum phomoideg 7 halt 14-9 Fuearium oxysporium 21 1406 601 gcopersici W ceghalosporium type 3 14.7 1+,8 ”41.9.25 globosus 10 11.8 5.1 £1223 hiemalis h 11.9 5.0 m sp. 3 1+.9 5.2 M sp. 18 14.9 6.3 W corjophilum 11 3.5 11.6 W oxalioum 2 11.5 11-9 2W pumurgenum 2 14.5 14.5 W sp. 1 14-0 LL00 W sp. 11 h.1 7.1 W nigricanjs 18 14.5 14.9 18%0222 sp. 2 1.7 5.0 W bulbicola 1 14.7 \Tpichoderma sp. 2 L3 1105 cc"Nu-ole 5 11-5 h-S \ 63 Discussion From the results of this experiment it is easy to under- stand how canners may be confused and baffled by various mold counts obtained by their analysts and by the Government. The fact that 0.1 percent visible Alternaria rot gave a Howard mold count of 57 in one instance and in another instance the same percentage visible rot, also produced by the same mold, gave a count of 11;. (Table v) demonstrates clearly the problem which may confront a processor. It is well known that it is difficult to determine the extent of visible rot with accur- aoy but. this is the task normally assigned to housewives on trimming belts in tomato processing factories. It is well known that ggllettotriohum phomoides may ”MU-1y decompose tomatoes given the proper conditions. How- ever, it was found frequently in this study that many tomatoes showing Colletotrichum phomoides lesions could not reasonably be considered as showing rot in the commonly accepted sense 01' the word. Even substantially increasing personnel on a trimming line in a factory could result in failure to remove 311 8“ch spots. when all visible rot is removed from tomatoes the “‘1de tomatoes themselves may show higher mold counts than tonlatcms which do not require trimming. It is apparent that M1detu... - Quail. .1111. I‘le b.l ' I. 1 - "VP, 611 unless the processor receives sound stock at his platform he is placed in an extremely precarious position regardless of expenditures for sorting, trimming, and mold count analysts. The hazards which may be presented, particularly by Colleto- trichum phomoi<'Jnc:1uded that pectin polygalacturonase did not appear to be a Varietal characteristic. Bell (1951) found substantially similar results, and suggested that the absence of the soften- ing eIIZyme in green tomatoes may account for the lack of dif- fiwlty experienced by industry in brining this commodity. l‘Ic(}olloch and Kertesz (1911.8, 1919) reported the presence in tOmatOes of a heat-resistant factor possibly an enzyme. 71 They called this depolymerase (or DP) and concluded that the loss of pectinic substances in processed tomato products is the result of pectinesterase (or PE) which first demethylates the pectinic acids and the DP which then depolymerizes the pectic acid substrate causing a loss of the colloidal proper- ties of the tomato product. MacGillivray and Ford (1928) presented a valuable dis- cussion of the contributions of the various regions, outer and inner wall, the inner locule tissue, the jelly-like pulp around the seeds and the skin of the tomato to its overall quality. Decomposition of Plant Tissues by Molds Most studies related to the action of fungi on plant tissues have been reported prior to 1920 (Kertesz, 1951). For many years plant pathologists have sought to determine the factors responsible for the development of parasitism in the molds. Branfoot (1929) ably reviewed the early research. Williaman (1927) also contributed an excellent review of the relationship of fungi to the enzymatic breakdown of Pectin in plants. Bate-Smith and Morris (1952) pointed out that each particular substrate under natural conditions seems to support only a limited microflora and to be al- most invariably attacked by this flora. They cited how 72 oranges are liable to attack by Penicillium digitatum which is quite distinct from P. expansum which attacks apples. Foster (19119) claimed the number of species of fungi known in 1938 was estimated at 89,000. According to Brown (l9h8) the invasion by facultative fungi consists of three stages, (a) prepenetration stage, (b) process of penetration, and (0) post penetration stage. He stated that no fungi have been known to secrete a sub- stance capable of dissolving the outer cuticularized surface of plants and in this same report he said that the act of penetration of plant tissue by fungi has been clearly demon- strated as being mechanical. He outlined how by the use of gelatin filters of graded hardness it had been shown that the molds, Botrytis cinerea, Penicillium glaucum and Rhizopug nigricans respectively had diminishing penetrating power. He offered this as explanation as to why Rhizopus nigricans has been found to enter only the least protected structures such as the soft fruits in contrast to g. cinerea and Rhizoctonia M which had been found able to enter most plants by direct penetration. Willaman gt £1. (19251 in studying the problem of the com- parative resistance of certain plum varieties to brown rdt em- phasized that the more resistant varieties had a tougher skin and a firmer flesh. Accordingly, they believed that resistance was due to mechanical resistance to the entrance of the fungus. 73 This was investigated further by Willaman (1926). The tough- ness of the skin was found to increase in all varieties of plums as ripeness progressed, the change becoming more marked in the resistant varieties. Rosenbaum and Sando (1920) reported a correlation between skin toughness and resistance to Microsporium tomato (Cook). Once inside the tissue the most outstanding feature shown has been that the cells of the tissue are disorganized in advance of the iflthldb hyphae (brown, 19h8) and pectinase secretion by the fungus Was mentioned by Brown (l9h8) as the nwst prominent agent of attack. In this report he claimed also that within limits a correlation has been detected be- tween parasitic vigor and the capacity to secrete pectinase into the culture medium. Many factors appear to influence the development of rot at this stage. Weurman (1952) found that there are inhibitors in pears which inhibit fungal PG but that their chemical na- ture and functions were not known. Internal resistance to the growth of most fungi was men- tioned by Brown (19h8) to be due to such substances as acids, tannins, ethereal oils, and glucosides and he indicated that the metabolism of the organism may also cause inhibition of growth as may desiccation of the tissue itself. In some cases aSumming reaction may occur or the formation of a cork bar- rier also may limit growth (Brown, l9h8). 7h Kertesz (1931) stressed that very little has been known abmnsthe discharge of enzymes secreted by molds into culture medium and pointed out that with changes in the medium, changes occur in the life cycle of the molds also, and that on account of different substrates the molds have produced (afferent amounts of enzyme. Also he said that while there lmve been many observations concerning the effect of various nutrients on the formation of PG the relationship has been far from clear. Menon (l93h) also showed data which indicated that the medium greatly influenced the precise behavior of the enzymes of any particular fungus. He claimed that some of its pro- Fmrties are greatly influenced by absorption from.the nutrient radium. Willaman (1927) suggested that the medium appeared to exercise a quantitative and possibly a qualitative effect 0n.the production of the enzyme. He suggested that the dis- solving of the middle lamella may be one of calcium removal. Kraght and Starr (1953) emphasized that any report that an organism does or does not produce PGv should be considered in the light of the medium and conditions. Fernando and Stevenson (1952) showed that‘ggtgytig'giggggg Spores which germinated on the cut surface of fresh untreated Potato tissue rarely produced any measurable attack. However, When the tissue was injected with water, definite attack 75 followed. They concluded that there was a factor associated with subturgid conditions in potato tissue which antagonized the action of pectinase enzyme of Bacillus cinerea but not that of Bacillus caretovorus. They believed that the close behavior of the organism.and enzyme to be a strong indication that the enzyme was in reality the active agent of parasitism. Gregg (1952) in additional experiments on the subject of injection or soaking, stated that while one might assume that soaking or injection would facilitate the diffusion of enzyme into the tissue, the water content of soaked and injected tissues was often very similar and yet these treatments gave rise to markedly different degrees of susceptibility. Holden (1950a) reported that in leaf tissue where pectic substances occurred in combination with calcium, the enzymes pectinesterase or PE and polygalacturonase or PG did not cause complete disintegration without removal of the calcium. She stated also that tobacco leaf fibre which had taken up the maximum calcium was scarcely attacked by PG even with prolonged incubation. Holden (1950b) believed that any differences in the ac- tion of various unfractionated fungal enzymes on fibre by Pbctkmn|10M and Enzyme 19AP (Rohm.and Haas), Botrytis cinerea, Aspergillus aureus, Penicillium expansum.and Penicillium.digi:, Egggm.likely was due to a difference in the relative amounts of the enzymes and not due to their presence or absence. ..I It Il‘lule . “lei Allin . -\ t. 76 Reid (1950a) reported that apple extracts were readily solubilized by the action of some molds whereas in black cur- rents the amount of enzyme resistant material was considerable. In.this same report he stated that the relative amounts of the enzymes in different preparations varied considerably, de- pending upon the strain of the organism and the condition of growth. The pectolytic action of organisms during decomposition of plant tissues has been a matter of concern to many workers. As early as 1929 Pitman and Cruess employed pectin solutions to investigate the hydrolytic action of microorganisms. 00133 (1926) reported the digestion of pectin by many microorganisms. Joslyn (1929) and Fabian and Johnson (1938) studied pickle softening reportedly caused by pectin splitting enzymes. Couchman (1939), during an investigation of the retting of flax, indicated that pectic materials definitely were decomposed. The liberation of enzymes by fungi is receiving increas- ing attention as more and more variables are recognized which cast doubt upon the results of investigations reported in the early literature (Sumner and Somers, l9h7; Smythe, 1951). Elrod (l9h2) urged that a clear distinction be made between the fermentation of pectin and macerating action and claimed that Bergey's manual (Bergey 32 51., 1939) has been confusing in this regard. Methods for examination of the pectic substances and en- zymes have been receiving increasing attention (McColloch, 77 1952; Jermyn and Tomkins, 1950; Reid, 1951b). However, there seems to have been little added to our knowledge concerning some of the basic factors governing the liberation of enzymes by fungi in many respects such as were investigated by Harter and Weimer (1921) and Weimer and Harter (1923a, b). Experimental and Results Production of Pectinase (polygalacturonase) by the Molds as Determined by Liquefaction of Pectate Gel The ability of organisms to liquefy pectate gel has been used by a number of workers (Miskeow and Fabian, 19533 Nortje and Vaughn, 1953) as a basis for separating pectolytic organ- isms. The Coliform Sub-Committee (l9h9) pointed out that the ability of an organism to liquefy a pectate gel has been com- monly related to its ability to cause soft rot in plants and listed two media containing sodium pectate. The advantages of pectate gel for the demonstration of the parasitic proper- ties of organisms dtté described also by Sabet (1951). One hundred and forty-five cultures of molds obtained from tomatoes during another phase of this study were cul- tured on the Northern Regional Laboratory sporulation medium (Gailey gt 51., 19h6). Spores and vegetative forms then were inoculated onto Petri plates of the pectate gel of Baier and Acre-‘- 78 a Manchester (l9h3) . This had the following composition: sodium hydroxide 8.0 ml Calgon 2.5 gm beef extract 3.0 gm peptone 5.0 gm sodium pectate 30 gm water to 1000 ml Observations to determine liquefaction and growth char- acteristics were made at 3, 8 and 30 days following incubation at room temperature. Organisms which produced no liquefaction at the end of 72 hours sometimes showed slight or pronounced liquefaction at the end of 8 or 30 days. The presence or absence of liquefaction of the pectate gel varied according to the genus, species and strain of the organism (Table XII). Marked liquefaction was produced by two of the two strains CK’Agpgrgillus agggg tested, all of the four strains of flgggg Eéfiflgng, all of the 16 strains of Penicilligm.coryophilum and all of the 19 strains of Rhizopgg nigricang employed. By contrast, only two of the l? cultures of Colletotrichum.pho- Heidgg produced definite liquefaction. No pronounced lique- faction was evidenced by most strains of'flggg£_globosus, Alter- m £21222, Oospora sp., Fusarium oxysporium, Rhizoctoni_a_ k “Preparation of this medium is described in detail (Cali- fbrnia Fruit Growers Exchange, Ontario, California, Research News Letter no. 20, reference no. 333). TABLE XII 79 PECTOLXTIC AND CELLULOLYTIC ACTIVITY PRODUCED BY THE MOLDS IN TOMATO JUICE Pectate Tomato Juice after 5 dgys Isolate Organism gel G-Z-f No. liquegy P Cellulasey H factio lug/ml rug/ml p 3 Alternaria solani None - - - 7 " " None - - - 8 9 " None - - - 9 " " None — - - 10 " " None - - - ll " ” None - - - 12 " " None - - - 16 " ” None - - - 17 " ” None - - - 5 " sp. None 1.1 ** 5.7 19 " " None * - - 21 Aspergillus clavatus None * 2.8 5.2 20 " niger +M 3e8 0.6 3e3 23 " +++ 1.6 5 .0 3 .1; 22 9 terreus + 0.1 ** h.8 2 1! none 8" “"3" 11.8 125 “ sp. None 0.1 0.1 u.7 25 Botrytis cinerea +++ - - _, 28 Colletotrichum phomoides ++ - .. - 30 ll -]7 + * None h.h 31 9 9 + - - - :7 Pectate gel liquefaction as detected visibly during the 30-day - period: + 8 slight, scarcely discernible; ++ = definite liquefaction but not complete; ++¢ = complete liquefaction of the gel in the plate. g/ Polygalacturonase expressed as Pectinol 10M. 3/ Cellulase expressed as Enzyme 10M. ‘ * Eggs than 0.05 mg/ml polygalacturonase expressed as Pectinol «a Less than 0.1 mg/ml cellulase (Cx) expressed as Enzyme l9. - 8 No determination made. 80 TABLE xII (Cont.) ” Pectate Imflate Organism gel Tomato Juice after 5Fdays No. lique- PG Cellulase faction mg/ml mg/ml PH 32 C 0 lie totri chum homoides None - - - 33 __T 2'1"— : 1.6 a 11.5 32 " ” None : I : 37 N I! NOno - .. .- 38 u n None _ .. - fig 3 2 None - - - kl : z + 0.1 as 7'2 (if; n it 4. f 2* )4: h5 : 2 + * None h.6 he a " + * None h.6 #7 " " None * *# h.6 hfl Iftlsarium oxysporium None * ** 5.2 1.19 " None 0 .7 M S 0 1 50 N ” None ‘n' fl 5 e 1 51 " " None 4N- “ %.6 52 fl " + 1e]. “'3“ e1 53 n " None 0.05 as u.9 5 " N None * ** 5.0 5 ” " None * ** h.9 58 " " None * ** h.8 59 " ” Nona it *5 11.6 20 n " None 0.05 ** 5.0 6:]?- fl " ‘0' “31' *‘31' (.Le7 " " None 0.7 0.2 h-% 23 " " None * ** h- 6% n " + a as 5.0 66 fl 3 * 41- 0e1 (4J6 67 2 n 11°” 0.7.05 :3 1'2 172 " cephalosporium - * ** NZ7 1 " None 0.05 ** h.8 13 N n + 0 e1 H (lea 76 ‘Fhacor lobosus None 0.7 ** h09 ;g “"‘W'E“W“" + 3.8 ** h.8 79 g : '0' 3 e8 *‘H‘ he 8 80 . None 3 e 8 M 5 0 0 II '1 ++ ZeS W (40 9 \\ TABLE x11 (Cont.) 81 “111:?“ Organism Pegzite Tomato Juice after Edayg lique- PG Cellulase faction rug/m1 rug/m1 pH 82 M‘ucor lobo 8 -—T LWJJL! + 106 w e 8% n " ++ 6.0 as t 3 87 n u + 3’8 ** “:8 89 " w * 6'0 M 5 O ' + Zes Non. 5.0 g; :: hiemalis +++ 2.5 ** 5 0 73 v" : +++ 1.6 as 520 7h " " +++ 2.5 None 5.0 bl _ +** 1.1 None h.9 ” sp. None " * None . gig {I u I 3'; fl {:02 - - - _ ‘ 0 w 5.2 0 - _.._ _ .. ._ ._ _ _... ._ ._ 31 M939- 32- + 0.5 w .9 92 n a + 1e]. *1“ 2.0 93 n n + 1'1 {Hi- 5'3 9h u u + 2'5 fl 6'2 32 ~ I 3'1 .. 5.. 97 z: z: + .2; «3” 2'8 98 n " None 0.5 None 5.h 199 ,, " None 1.1 0.1 6:3 133 a " £23: 2.5 gone h.3 . 6 0 102 ” r + 3.8 None . i0): 2 ': None 0.2 N23: £313. 135 " " None 1% No” ’0 106 9 n + ; §* 6.1 107 F " + * one 5.0 _ _. _' " 0 None 1.6 :: 22g 108 " -' " " " " " -' -"- '- '- .— ._ ._ 109 Penicillium coryophilum «H 0.5 o 6 6 110 " +++ 1 6 ° h' 112 q " ++* . 1.0 u.1 1H; t d +++ 0'3 1'6 3’5 . a . 0.5 2.8 .6 115 - h 1r? n u *** 1.6 2.8 u. w u +++ 0,? 1.0 3.% +++ 2.; 2.8 u.2 \\ 82 TABLE XII (Cont.) Iaolate No - Organi em P0613?” Tomat 113:6_ o Juice after 5 days faction [£91111 Cell 1336 121 Fe 1 1 2 ml pH 122 n cillium cor o hilum +++ 1 1 .1 13?. :: ~ 1:: °-7 is M I! 0 S . . + . 1.0 m ,, 0.7 1.6 32) 116 " oxalicum . ++ 6 O S 113 n N ++ 1.1 1:8 fi.g 119 " Eureurogenum None * N . N one .320 ” 8p one * None ti; - _- '_ ‘- , +4 ‘_ ‘_ ‘_ _. _"- ‘— 0.7 2 8 12 - - - - . 123 Rhizoctonia sp. - - - - — - [to 130 II n None * 135 .. * 77 7'1 138 n n A one * Lne L.- e I lho " " None * None u.7 lul " " None * fione 5.0 | Non one u 6 m3 3 " a * . " None None u.6 HM- " " None 3.1 ** h-3 - ~ - - - None None 0.3 1.1-.5 11:8 hizoaue nigricane - _ - _ — _ - - - ufi + 0.1 150 ". ' Hut 0 1 M “.7 l n u + . fl . lg; n .. H" 2.85 ** fig 15 n " +++ 1 . 6 None ‘4. e 9 }5 '.' " I 012 0*: 1M5 n . . 155 oi I: ++ 0.1 M has 13; a + 0.05 ** fi.; M o 158 .. .. *” 3.35 H L“? {23 .. .. .. .1 “.2 "'7 n , ++ . 1.1 z: ++ 0’05 .. H 163 .. " +++ 0.1 W 5.1 :22» :' 1:” 3 ”'7 y [t 4% 0.1 “.7 u + w u 6 N ++ 0.2 ** . 0.1 ** h-S h-S 83 TABLE XII (Cont. .) -_—— Iafilate Organism 2.22:6. Tomato Juice after 5 day; °’ lique- PG Cellulase pH faction tug/ml rug/m1 SS Rhizogus nigricane ++ - .. .. 162 n sp. +++ 0.6.5 ea. 5.0 165 " " None 0 . 5 M 1+ .7 167 Stilbella bulbicola None 4:- t-n- 11.7 168 Trichoderma lignorum None None #11- 14.5 169 None None N 11.3 ‘. r I. 5' - tn DA “1 . -- (tr ‘1 (l‘ M ‘u .1. g I ‘1' 8b, solani, Stilbella bulbicola, Trichoderma lignorum or Penicil~ _l_i_u_m purpogenum. The cultures which previously had been the most active in breaking down tomato tissue were among the most active ones in the liquefaction of the pectate gel, as for example, Asgergillus ni er, isolate no. 23 and Rhizogus niger, isolate ' _.,, rust: -’ no. 150. The majority of the organisms which showed no well- marked liquefaction of the pectate gel were those which had 9 "I“ ‘5 -('( A y' 9 not produced severe symptoms in tomato fruit within a short / Li time after inoculation. This applied particularly to Alter- Egg; sp. and Colletotrichum sp. This test was most satisfactory in indicating the organ- ismswhich were actively pectolytic. It was difficult to employ this test for separating organisms that only slightly liquefied pectate gel from those organisms which showed no activity. Sometimes slight lique- faction appeared under mycelial mats and this liquefaction was masked by the growth of organisms and was very difficult to discern. It was observed, too, slight liquefaction occurred only at the edge of some colonies. This was frequently noted in the Petri plates containing Colletotrichum sp. The amount of aerial hyphae produced by the molds was not related to the behavior of these molds in liquefying pectate 891- The top plate of Figure 5 shows a Petri plate laden with hyphae produced by Mucor globosus. Thisuorganism did not :i.:e 1 .2 eat ‘a a "‘ . .1. n .u i D.- .. 5. st. ‘I‘s. '! -..e. 85 produce visible liquefaction of the gel. The small section of medium at the t0p of the plate became detached when the gel was pried loose to determine if liquefaction occurred under the mat. The plate at the bottom of Figure 5 shows Aspergillus up. With almost an absence of aerial hyphae but liquefaction 01' the gel. when the plate was placed vertically the medium “1:. i I ran to the bottom of the Petri plate. Two strains of Fusarium .: J, ogsporium are shown in Figure 6. One had a considerable amount of aerial hyphae, the other had almost none. Neither 1 J culture produced liquefaction of the pectate gel. A j ‘ Hany differences in the colony characteristics of the various genera were apparent (Figures 7 and 8). The two strains of glternaria solani in Figure 7 showed little resem- blance to each other. Production of Polygalacturonase and Cellulase (Oz) by the holds in Tomato Juice as Determined by Cup-plate 588333 The present investigation was undertaken to obtain an estimate of the relative amounts of polygalacturonase and “nulase produced in tomato Juice by the molds isolated. v1seometric assays have proven very satisfactory for p°°t1¢ enzymes studies (Reid, 1950a}. Tests employing pec- tin for meaguring the combined PG and PE activity of fungal ”tact” have been used by a nuinber ofworkers (Bell 215, 2.1.” 19503 Bell, 1951; Phaff, 19M; Luh and Phaff, 1951: White and '86 .F 13- 5 Pectate gel liquefaction experiments illustrated by Aspergillus sp. and Mucor globosus. Lower plate: Asger~illus sp., medium was liquid and detached from edge of ketri plate. Upper plate: "ucor qlobosus, medium was solid, not“detached from edge of Tetri plate. 87 Fig. 6. Comparison of the aerial wcelia produced by two strains of Fusarium ogsporium. 88 "R... p r H. . *yd-‘fi ‘ 1"“- N-,..v--2 m-r'.‘ Fig'77- Differences in the growth characteristics of two strains of ~‘ig Alternaria solani cultured on pectate gel medium. 89 ~Fig-8o Differences in the growth characteristics of four strains of Colletotrichum phomoj des cultured on pectate gel medium. 90 Fabian. 1953; Beneke gt 2;" 1951;; Reid, 1951a). For deter- mining P.G activity alone, viscometric assays employing sodium pectate have been conducted by Reid (1951a) and Nortje and Vaughn (1953). However, some types of viscometers, particularly Ostwnlds, are cumbersome to use and errors may result if minute particles are present in the preparations (Kertesz and Loconti, 191414.) . A criticism of methods was made by Pandhi (1953). i'l'he method selected was essentially that of Reid (1950b) and Dingle 9.2 3;. (1953), a cup-plate procedure which has been used successfully for screening preparations for PG, cellulase (Ox) and other enzymes (Dingle and Solomons, 1951, 1952). It "as not the intention ofithis study to measure small amounts of PG which might be produced by some of the molds or that PG Which might be present normally in the tissue of some tomato fruitg. (Kertesz, 1938; MacDonnell 333., 19,453 8011, 1951)- Briefly, the method selected consisted of placing minute munts of the preparations being tested into cups which were °ut 111 the medium. After incubation with controls the plates we" flooded with a reagent, readings made and the amount 01' enzyme activity present determined from a standard curve. I h the details of the method have not been readily available they are given here. I“-“or the PG assay, the medium consisted of 5 grams ammonium oxalate (to remove any calcium present), 10 grams sodium pec- * s ta)“- 0.1 gram salicylanilide (to prevent mold growth), \ .l 0n “Sodium polypectate, obtained from Sunkist Growers, tar10. California. 91 20 grams agar, brought to 1 liter with potassium dihydrogen phosphate buffer, pH 14.5. The reagent was SN hydrochloric acid and the standard was Pectinol 10.” For the cellulase assay the medium consisted of 10 grams sodium carboxylmethyl- cellulose, 0.1 gram salicylanilide, 20 grams agar, brought to 1 liter with Nalpole's acetate buffer pH 11.5 (Hawk 9_t §_1_., 1951). The reagent was a 10 percent solution. of copper acetate. Pectinol 1014 was employed as a standard for measuring both PG and cellulase by Dingle and Solomons (1952). Unlike PG, the cellulase content of Pectinol 101“! has not been standardized from batch to batch (Labbee, 1951+), whereas Enzyme 19“ has been standardized for cellulase content and therefore was employed in this study. Both standards were kept at 5°C under silica gel. Specially constructed stainless steel and glass frames heWing an internal surface area of 6 1/2" x 12 1/2" were made to hold the media. To conduct an assay the glass plates were "199d with ethanol and warmed by placing in an incubator at 60°C. Plates were poured by adding a standard amount of the “dim at 60°C to provide a L; m layer. A spirit level was “N1 to check‘even distribution of the medium. The medium was Pal-Pad on part of the plate with the minimum of tilting and bubbles were broken with a hot needle. When the agar was set (Senerally 30 minutes) a paper template was placed under the \ d “Pectinol 1014 obtained from Rohm and Haas Company, Phila- °lph1a, Pa. Enzyme 19 obtained from the same source. -,v 92 glass and cups 8 mm in diameter were made in the agar with a sharp cork borer. Those agar discs were removed by a diamond shaped needle. The center of the holes were approximately )4. cm apart, Accurate amounts of each preparation under test were added to duplicate cups employing a rubber bulb attached to a Pifice cut from the end of a capillary pipette, the glass or Which had been drawn out to provide delivery of 0.06 ml of the preparation under test. Between tests the filler was rinsed with pH 7.0 buffer and distilled water. Duplicate cups containing four logarithmic concentrations of the control were included on each plate. These control concentrations were Prepared with tomato Juice inanediately preceeding an assay. The plates were covered with aluminum foil to prevent evaporation and placed in a 37°C incubator. After 18 hours “1° Plates were renn ved, flooded with‘t usagent and the readings made. A BI‘ownlee 33 2.3.10 (1914.8) recommended that in testing pre- parations the cup-plate zones be magnified. A Delineascope apparatus provided with an accessory stage rackto hold the 91‘“ Proved very satisfactory (Figure 9). This was arranged 5° than: segments of plates were magnified Iii times and pro- J°°t°d onto a grid. In the PG assay, the diameter of the sharp. inner area of hydrolysis was measured (Dingle 213, 9}.” 1953). In the cellulase assay the outer zonefdiameter was mameddirectly from the plates with calipers. Fig. 9e Modified Delineascope upgwratus employed to msqnlfv in the estimation of poly 'licturnn:w= notwity ;1.;. extracts and cultures. ' 93 “A )1. A -. + v\. , . 9h To determine the sensitivity of the tests logarithmic dilutions were made of a b, percent solution of Pectinol 10M in tomato juice which was placed in quadruplicate cups in a plate which was then incubated and treated in the manner described previously. Standard curves were prepared by Plotting the zone diameters against the enzyme concentration (Figure 10). Readings were made directly from these standard curves during subsequent sample tests. One hundred and twenty of the molds employed elsewhere in this study were transferred to the sporulation medium.(see Galley et al., 19116) and incubated for one week. Then heavy inocula of spore and vegetative cells of the moldswere added to small flasks containing 100 ml quantities of tomato juice. These flasks were left in standing culture for five days. The liquids under the mycelial mats were removed, adjusted to pH LS and assayed for PG and cellulase (0x) using the method just described. I Pronounced differences in the production of PG by the individual molds were apparent (Figure 11). A photograph of ‘ tifpical magnified section which was projected onto the grid is shown in Figure 12. The results are given in Table In (Page 79 ). Aawas to be expected from the resultsob- t‘imd earlier in this investigation, most of the prepara- tion: showed an increase in pH value due to the presence of IHolds. with few exceptions, little or no PG activity was shown in the tomato Juice samples containing Alternaria M, 95 E , l g. l “ decreasing” logarithmic concentrations of mold polygalacturonase as shown by the cup-plate assay (Pectinol 10H). *top to bottom 96 Fig“ 11. Variation in polygalacturonase potency produced by 1h different molds in tomato juice. Duplicate assays of each extract were placed horizontally. The bottom two rows were controls of standard rectinol 10? arranged vertically. Fig. 12. Cup-plate zones magnified on grid showing measurable polygalacturonase activity in three tomato extracts. No measurable activity in one sample. I l/h. 97 to .y- 98 lelietotrighum phomoides, Aspergillus clavatus, Aspergillus terreus, Fusarium oxysporium, Euiarium cephalosporium, Rhizoc- _t_c_>r_1_ig sp., Stilbella bulbicola and Trichcgerma lignorum. This was in contrast to considerable PG activity which was shown in samples inoculated with M sp., Oospora sp. and Rhizopug sp., the molds which had been the most active in attacking tomatoes under experimental conditions. The test for cellulase also proved satisfactory and the differences in the production of this enzyme by the various molds were readily discernible (Figure 13). or the molds tested, all cultures or'renicillium coryophi1um and W M were unique in producing cellulase con- sistently and at a high level. Strains of Penicillium coryophilum produced this enzyme in appreciably greater quantities than any of the other molds studied. H329; globosus and 11199}; hiemalis and Mg sp. were shown t0 Produce a high titre of polygalacturonase in tomato juice but a 10" cellulase titre. This was noted also in respect to Oospora 39° These same strains of 31299}; lobosus, N223; sp. and Oospora sp. hm shown no liquefaction of pectate gel. MOat of the organisms produced cellulase but only in small mun“. (Table III). There appeared to be no correlation between cellulase production and PG production by the molds. 99 Fi g. 13- Variation in cellulase (Cx) potency produced by lb different molds in tomato juice. a Duplicate assays of each extract were placed horizontally The two bottom rows were controls of standard Enzyme 19 arranged vertically. 100 Polygalacturonase Concentrations in Partially and Wholly Decomposed Areas Trimmed from Tomatoes Tomatoes which showed macroscopic evidence of mold growth were collected from fields in Indiana and Ohio as outlined elsewhere. At the site of each mold invasion a 10 gram quantity of the visibly affected area was cut out with a scalpel. To this was added a small amount of two percent saline solution and the mixture was ground in a Waring blender. The pH was deter- mined with a Beckman model G pH meter. One mixture was adjusted to PH 6.0 with NaOH, the other. was left at the pH level noted. Each preparation was filtered through cheesecloth and brought to a volume of 50 ml with saline, poured into a test tube. layered with toluene and held at -lO°C until examined. This method was essentially the same as that given by Bell 2E .93.- (195U. The samples were examined for PG activity by the cup- plate method described previously. R08111ts are given in Table XIII. Only 11 of the 208 samples °r wh°11y or partially decomposed tissue trimmed from tomatoes ”hm“ concentrations of PG in excess of 0.05 mg/ml compared with an arbitrary strength of Pectinol lOM. Greater activity was noted only in samples from which M, Oospora and Rhizopus had “911 isolated. No significant differences were found in the PG content of samples which had not been adjusted to pH 6.0 as cornp'flred ‘50 3d- 11 . J Sted 8amples. Generally, the extracts which showed high con- ce ntrations of PG also showed low pH values. TABLE XIII 101 POLYGALAGTURONASE CONCENTRATION AND pH OF PARTIALLY AND WHOLLY DECOMPOSED AREAS TRII‘MED FROM TOMATOES 8:121:10 characteristics of Cut-out Area pH P3113] Organism Isolated 9a soft, brown 6.1+ None Rhizoctonia solani 9c soft, overripe, green and black 5.2 None Alternaria, Clado- growth sporium, Rhizopug 9d soft, yellow and green rings 6.1 None Rhizoctonia 96 firm, black growth in splits h.7 None Alternaria 9f soft, sunken spots, brown 1+.9 None Colletotrich_u_m ml. phomoides 98 soft, greenish yellow 6.1+ None Rhizoctonia and bacteria 9h soft, white aerial growth 8.14. None Fusarium 91 soft, greenish 5.2 None Rhizoctonia, some Mucor 91 firm, sunken, shrivelled, black 5.7 «- Mucor, Rhizopus Ilternaria and ii usarium on surface ‘3" e :3: soft, pinkish white hyphae 7.0 None Fusarium 10 sort, pinkish white hyphae 8.1+ None usarium 10; 80ft, sunken spots, brown li.S None Colletotricrgm 30ft 7.7 None Fusarium with 100 with bacteria 10: Sort spots, sunken, twelve spots 6.? None Colletotrichum 30ft spots, sunken, dark, six 5.3 *- oliggjtotrichum 8pots i851 firm, sunken, black spots, four 5.7 None Colletotrichum 10i 80ft area, 1" diameter 6.5 None usarium 30ft spots 1'.‘ diameter (:1 surfhce 5.11. None Oolletotrichum on surface, some 101 Fusarium 33:1; spots, sunken, concentric h..9 «n- Colletotrichum 1138 fig 30ft: area 2" diameter 7.0 None Rhizoctonia 11c 3‘31“: spot 1'? diameter, dark 5.3 None Ilternaria 11d “rt spot 1/14." diameter Li.5 fl- finicillium 119 Sort, decomposed thi ‘5 Mucor 11: soft, black spot 7.6 None Fusarium, Alternaria 80ft, overripe, 1/2" crack ll.8 None EnizopusI some . Oos ora, Mucor on 118 801‘ surface t 706 None Fusarium 88 than 0 e05 Wgalacturonase expressed as Pectinol 101?. mg/ml polygalacturonase expressed as Pectinol 10M. \\ new. x111 (Cont .) “_- 102 L. _‘1 ‘_:_‘_7 Sample Characteristics of Cut-out Area pH PG Organism Isolated N00 mg/ml 111“ 3 Spots, 3/ " diameter 14.9 * Rhizopug, Oospora, _ yeast lli insect injury entirely soft 6.14. 11- Penicillium, Oospora, Alternaria 11.1 soft spot 3/1i" diameter, black 1i.8 * Alternaria, Rhizoctonia 12s soft area entirely 5.1 w- Fu arium, yeast “"‘ ._ 12b dried, black surface on soft 7.8 None Oolletotrichum omato 120 very soft, a split 5.5 it Oospora 12d black spot 21-1/2" diameter 6.1 None Alternaria 12° soft, water-soaked areas 6.1;. None Rhizoctonia 121‘ soft, dry 7.6 None Fusarium, Oospora on surface 138 soft, greenish yellow 1/2" diam. 6.14, «- Rhizoctonia “WWW; 211 soft spot, 1" diameter 5.0 it Oospora ' 121 broken skin- 12 30ft, decomposed 7.2 None Rhizoctonia 1 3 spots, 1/2" diameter h.5 a» Oolletotrichum 3: soft, water soaked 14.5 i Rhizoctonia 13° firm, black spots, green edges 3.5 iI- hizoctonia 13:! 30ft, brownish rings 5.0 0.05 Rhizoctonia 13. 30ft, brown spot, 2" diameter 7.1 None Ehizoctonia 131, 80ft, greenish 7.5 None Rhizoctonia, yeast 13 30ft, skin broken 6.1 4;. Huger, Oospora 13% 3°31? 6.6 {I- F‘ zoctonfa 131 30ft, grayish 6.0 None Rh.zoctonia 131 soft, greenish 7.0 None mLzoctonia, yeast “Pots l/li-l/Z" diameter 14.7 None OolletotrichLm fl; 80ft, brownish spot 2" diameter 7.8 None Fusarium sunken, tough tissue 6.8 None F'usarium, Alternaria, 111C da Mucor rk. sunken, shallow spots 6.8 None Fusarium, some 52.4.2.2: 111d fi 223 on surface m. rm. black, sunken spot 1i.3 None Alternaria, W 1hr soft. 1/2" dark, watery 1i.6 «I- w . lug “ft. black spot 5.1... «n- Mucor, bacteria watery spot, dark with green 5.1.. None F'usarium, Mucor m1 °°nters . 11” 3113110", sunken spot, 3/14" diam. 14.7 None “106118. a110w, brownish black spots 14.5 None 3312223..“- 15a 81mllow, black cracks 1i.5 None Fusarium. Some Alter- 15b .naria on surface ahallow, 1" diameter spot, dry 6.2 None Alternaria, Fusarium \\ ‘~.-..._... _- TABLE XIII (Cont.) L t 103 3;?“ Characteristics of Cut-Out Area pI-I PG Organism Isolated 15c shallow, 1-1/2" sunken spot lull 4* Rhizoctonia. Some . Oos ora on surface 15d dry, brownish black 3/14" [p6 None AIternaria diameter, shallow .. 156 soft, brown split 6.14. None Rhizoctonia 15f 80ft, brownish, watery 5.5 None Rhizoctonia. Some A ternaria on surface. 153 80ft, brownish 5.5 None Rhizoctonia. Some Stilbelg on surface 15h soft, dark brown rot 1+.0 * Rhizoctonia 151 dark, brownish black rot 5.3 * os ora. Some Muco , 1 §§EIbela on surface 55 b1§ck, watery rot, foul odor 3.5 0.05 Mucor, Oospora 16‘ tough, leathery, black growth 1.1..8 None Rhizoctonia 16b 1‘I‘Qm 1" diameter spot Burlken spot, completely rotten 5.0 *- Rhizoctonia, Alter- naria 16° 8hallow, watery, brown spot 1" 1»? 0.05 Rhizoctonia, Oospora 16:: diameter, insect hole . lbs all black soft rot, watery 5.2 il- Mucor, Oospora all soft, from wide crack showing 14.3 0.2 Eizoctonia, Oospora 16: Blimy growth, color normal 1-l/.2" soft brown area, showing 14.8 41- Oospora 163 concentric rings, split 16h 8\leerficial spots, light brown ling None M celia aafierficial black, sunken 3/14." 14. None Colletotrichgg ameter 161 shallow, 1" diameter black spot 6.1 None Fuaarinufl. 163 Pink surface mold completely rotten area from 5.1 0.05 Oospora, Alternaria ril-‘uit eaten by hoppers, covered “1 th mold E: £1l]. black rot 6.7 «It Rhizoctonia 17:: all soft rot from 2" area 6.14. None Rhizoctonia Vex-y little rot, soft from 14.6 None IIternarIa, Oospora 17d t'kjinsect injury 17. in layer of rot in crevice 14.5 0.1 Mucog 171‘ all dark brown soft rot 5.0 None usarium, bacteria .ama-ll proportion of rot, soft 14..1+ .* Eucor, Oospora 17g acOOmpanying insect injury _ . all granular rot, from cracks 7.5 None Fusarium 8hCriwing orange mold and . .171' 8hI'ivelling sort rot from insect injury h.Li it Mucor, Oospora TABLE XIII (Cont.) 1014 Sfimple Characteristics of Cut-out Area pH PG Organism Isolated °' . me/ml 17g all granular rot, color normal 7.5 None Fusarium 1711 only a film of visible rot, 6.8 None Fusarium balance slightly soft from cracks 171 all green rot, from 1" round 7.6 None Fusarium soft spot covered with yellow mold 17J thin scab from cracks on surface h.5 None Alternaria 17k all dark rot from a 3” area 1i.5 None Rhizoctonia, Oospora containing a crack . 171 thin scab from 3/1i" soft rot spot 5.2 None Colletotrichum 171:: all soft rot from wide crack 1i.6 «- ospora across 1/2 of fruit, creamy growth 17“ thin scab from an area eaten 1.]..0 * Penicillium, Alter- away by insects naria 17° thin scab from a dry sunken 14.9 None Alternaria spot 1" diameter 17p very little rot from a shallow Il.7 fl- Fusarium soft spot l-l/Z" diameter 17‘! very little rot from a 1" 1i.3 0.1 Oospora, some 1 diameter insect injury . Rhizgpus on surface 7r all soft rot, color normal from 7.14, None leletotrichLm, 17 l/2 of soft tomato Rhizopus ' tough, soft rot from cracked 7.2 None Fusarium, Alternaria 17t fruit pulpy, decayed tissue from black 7.1 None cracks i3: all soft rot, red 1i.8 None Fusarium, Alternaria 18 all soft granular rot 7.2 None sar um, ternar a 183 all soft, black rot 7.2 None Colletotrichum all soft, dark rot from fruit 5.9 None Fusarium. Rhizoctonia 18. showing crack soft, mushy tissue from fruit 3.9 0.3 Rhizopus 181' with cracks 80ft tissue from a firm brown 1;. None Colletotrichum 13‘ area 80ft spot 3" diameter 5.3 None Fusarium, Colleto- 18h 'tPIChum' 181 8Oft, dark rot from a dry, black 6.2 Bpot with surrounding soft area yellow, small brown spot itch None Alternaria, .Oolletotrichmn None C Iletotrichum, Altgrnaria 105 TABLE XIII (Cont.) M “3:. mph Characteristics of Cut-out Area pH PG Organism Isolated ' . mg/ml None Alternaria, Igolletotrichgm f’ ;r 18 j dark, tough rot 19a dark, soft rot 5.8 None Rhizoctonia 19b all dark soft rot 7.2 # Rhizoctonia 190 all soft rot, greenish 7.0 0.05 Rhizoctonia 19d all soft rot, orangish brown 7.6 None usar um 190 all dark dry rot h.u * Fusarium. 0n surface Alternaria 19f 1/2 dark rot, l/2 normal tissue h.h None AIternarig, Fusarium from cracks showing black growth 138 all firm brown rot 6.9 * Fusarium 19h all soft brown tissue 8.2 None Fusarium 191 1/2 hard black rot, 1/2 soft 6. None Pusarium brown rot 193 'brownish black rot 8.0 None Fusarium, Oospora 20a thin surface scab 1i.0 None Ag; er illus, hizocton}; 20b soft rot, dark 5.7 None Rhizoctonia. Some Fusarium on surface a” all rot, soft 6.9 None Mucor, Rhizoctonia 20d soft tissue h.2 None Yeast. Bacteria on 2 surface - 0° Ininute black spot .h.3 None Alternaria 20f dark brown mottled surface spot 14.3 None ficteria. Fusarium 20 on surface 20% all brown rot 6.9 None Rhizoctonia 201 slightly soft tissue from cracks &.9 0.05 maze us small amount of black rot from .8 None Ilternaria cracks 3811‘ small amount of black tissue h.l None Alternaria, Fusarium 1/3 of sample rotten 1l.2 None Ilternaria, F‘Esarium 21‘ brown rot f , oul odor 6.1 None Oos ora, thor 3%: heavy black rot 6.7 None Fisarium 21d &11 white rot 7.3 None F'usarium, bacteria 21, :gjft, foul odor 1 .1 2.3 303; lgucor, F‘ufiarium te rot in insect n ury . . os ora, ucor 3}; 8mall amount of black tissue 6.5 None IIternaria, F'usarium 211 all'lall amount of black tissue 14.7 None usarium . 213 dry, hard rot 7.0 _* Fusarium dark rot 6.5 None F'usarium, yeast H g-w 1:: Ind-h..._"-. .__ TABLE XIII (Cont.) I:‘ ——‘-2_ 106 SW19 Characteristics of Cut-out Area pH PG Organism Isolated No. tog/ml . 22s soft, slightly brown color 5.0 None Rhizo us, Mucor 22b 3/14. rotten section 6.6 None Alternaria 22c dark, pulpy black rot 7.7 0.05 Alternari; 22d 1/2 soft red tissue 5.5 None lternaria, bacteria n surface some fihizoctonia 22a 1/ 2 brown rot gm None Colletotrichum 321‘ all brown, firm rot .0 None izoctonia 223 2/3 dry, black rot 11.8 None Alternaria 2211 all dark brown, firm rot 5.2 «- Rhizoctonia 221 3/L4 soft, brown rot 5. None Rh. zocton a 22.1 all soft, red rot 14.2 0.3 Rhizopus, yeast 23‘ black, hard rot 5.3 None Alternaria 23b 3/14. soft, red rot 14.8 0.05 Mucor 25° all firm, black rot 7.2 None Alternaria 23d all soft, red rot 5.6 1.1 Mucor 23° 1/2 dry black brown rot 5.1 None Ilterparia 23f 8.ZLZI. soft, red rot 5.0 None 233 all soft red rot 14.8 N- Bhizoctonia 23h all soft red rot 14.8 {- Rhizoctonia 231 all soft red rot 14.7 0.3 Mucor EM Buntill amount of black tissue h.6 None Alternaria aw- lv/JB black tissue h.8 None Alternaria Zhb 3 soft tissue, some small 14.7 None Oolletotrichum sIllnken spots 2211:: 1/2 brown rot 5.0 fl- Colletotrichum all soft red tissue 14.1 it Oolletotrichum, a” bacteria 2111' all soft brown tissue 6.6 None Alternaria 1/2 dry black tissue 5.5 0.05 Fusarium. Some Oospora 2&3 on surface 21-1-11 a1l dry, black rot 5.7 None ggizoctonia 21d all soft red rot 5.6 s- Oospora, Rhizopus 21” ‘11 soft, red tissue 6.9 None olletotrichujm 1/3 dry, black rot 5.2 None Rhizopus 2 2%; ail dry, black rot 5.6 {- Alternaria 25. 31V 2 dry, black rot gs; None ternaria /2 soft red tissue, 1/2 black .9 None _ 25d tissue 25s greenish orange rot 14.9 - all soft red rot 5.0 None Colletotrichum, yeast \\ 107 TABLE XIII (Cont.) Sample Characteristics of Cut-out Area pH PG Organism Isolated mg/ ml None Rhizoctonia § Rhizoctonia None Colletotrichum. Surface ggrmoden- drum, Alternaria 25! 3/14. soft brown tissue 253 all soft brown rot 25h l/2 soft red rot Y‘f’f‘ 0 C120) 251 all firm black rot . * Alternaria 251 all soft, brown rot . None Mucor, Rhizoctonia 26a all soft, dry rot . None Alternaria 26b soft tissue . None Fusarium 26c soft spot . «- Rhizoctonia 26d soft, tissue . None Fusarium None Ilternaria None Rhizoctonia 26° f‘ in, black tissue 261‘ all soft brownish red rot 26% soft, brownish red tissue . None Rhizoctonia 261‘ soft, brownish red tissue . None Rhizoctonia 261 soft, dark red tissue with black . None fiber-naria, bacteria 6 spots 2 3 firm, black tissue . None Alternaria 27a soft, mushy tissue, red . None Oospora 27b soft, grayish black tissue . fi- Oos ora 27c soft, light brown tissue, None Colletotrichum 9 shallow spots 37d soft, light brown tissue from None Colletotrichum 27 a timed area of spots ° 80ft tissue . *- M celia fl- $1 zopus None Alternaria :3]: Soft tissue 3 a~11 black tissue 0 F'HOONUNN U N (DOW-F 0 WWW F Ulw-F'HF’WCLHUI WO‘ 27h al . 1 soft rot . 0.3 RhiZO us. Mucor on surface 271 Water soaked tissue . None Colletotricrgm. 2 Oospora 7‘1 all dark red rot, soft . fl- Rhizoctonia 28; None Phythophthora None .hythophthora None Phythophthora None flhythophthora None gPhythophthora None Phythophtnora None flhythoghthora None Phythophthora 28b 8li'lall amount soft tissue 28c arrtall amount soft tissue 28d 8(bfft tissue from 2" spot 23, 80ft tissue from small spot 28: all soft tissue 283 all soft tissue 26}; all soft tissue, green fruit a‘ll soft tissue, green fruit \ UlO‘O‘O‘UlVIU'lU'l \J'l U‘l 43‘0\F'U1 U'l O‘F-F' ‘1 O‘U'lU'lmUlU'l-P'O‘N U10 108 Discussion In Table XII it will be seen that the molds which did not lJmnaefy pectate gel generally did not produce a high titre of 1%} in.tomato Juice and that this was below 0.05 mg/ml. However, in some instances when no liquefaction of pectate gel was ap- Parent as, for example, by 14329.2. globosus and Oospora sp. the concentration of £6 produced by such molds in tomato Juice was substantial. 'A comparison of the pectolytic activity as shown on both media would not be eXpected to show identical re- sults. The lack of agreement between the pectate gel liquefaction tesrt and the cup-plate assay could be attributed to many fac- tors. The two media were not identical. Even minor differences in the medium on which fungi are grown have been reported to influence enzyme production by fungi (Kertesz, 1931; Manon, 1931+). Also, changes in the pH of the media as the molds developed may have influenced the concentration of the enzymes present (Fernando, 1937). It has been suggested that proteolytic Qnqunes produced by fungi may destroy PG (Luh and Phaff, 1951). {Furthermore, one should not overlook-the appreciable A time factor involved in the testing of the organisms and in the sample storage. In the pectate gel tests the organisms were in contact with the medium for a period of thirty days, mills in the tomato Juice there was a period of five days. 109 The density of the sowing of the spores and also the number of spores which developed on the media (Brown, 1917) also might have had an influence on the concentration of en- Izyme produced. Results from the cup-plate assay technique for the es- timation of PG has been shown to coincide with a loss of col- loidal properties and to viscosity measurements (Dingle _e_t El” 1953). The test for liquefaction of pectate gel is based on a loss of colloidal properties ’Coliform Sub-committee, 19h9). Only one reference was found concerning the application 01' the pectate gel medium in determining the pectolytic prOper- ties of molds and this made mention of only two mold cultures (Misekow and Fabian, 1953). The present study shows that the liquefaction of pectate 881 by some molds may require a considerable period of time and that some liquefaction may be masked by mycelia. This study indicates that the molds isolated from tomato fr‘uits vary in their ability to produce pectolytic and cellu- 1"liftic enzymes. The cup-plate assay while more intricate than the pectate 3‘31 test was very satisfactory in showing these variations and was free from the errors associated with viscometric teats. Representatives of the genera, Mucor, Oospora and Rhizopus When inoculated experimentally into tomato juice produced an ap":~’-"-"eciably higher concentration of PG than did all of the 110 other organisms tested. It is interesting to note that of the many areas trimmed from partially and decomposed tomatoes, only those tomatoes from which £12293, Oospora and Rhizopus had been isolated also contained a high PG concentration. A loss in pectic constituents in tomatoes has been reported to coincide with softening and deterioration of this fruit (Le Crone and Haber, 1933). Dryden _e_t; 5;. (1952) on the basis or a study of apple puinice‘similarly believed that it was the decomposition of pectin which produced rot in apples. Ker- tesz and Loconti (19M) similarly reported that commercial pectinase caused a decrease in the gross viscosity of tomato Juice solids. Obviously the tests employed in the present study were effective in indicating the pectolytic activity 01' the molds and the production of PG by these molds correlated with the severity of their attack of tomato fruits under ex- Perimental conditions. Tests for galacturonic acid as an in- dicator of decomposition have not been successful (Almendinger it; $1., 1951;). A possible explanation for this may be the utilization of galacturonic acid by the molds themselves (Kraght and Starr, 1953). The results of Dingle and Solomons (1952) showed that’Cx was Produced in appreciable quantities by a high percentage of the organisms tested. The production of great measurable collullase activity in the present study appears to have been d°p§ndent on the genera and species (Table XII). A discussion of the cellulase production of microorganisms will not be 111 entered into. This is well covered in the literature (Fuller and Norman, 1914.5; Saunders ,_e_t_‘. al., 19116; Elwyn g3; a_._l., 1950; Reese _e_t al., 1950). It would be very desirable to investigate pure enzymes preparations many of which have been made available only during recent years (Holden, 1950b; Scheffer and walker, . 1953 ) . 112 GENERAL DISCUSSION One could not have anticipated the many types of molds which were found in Indiana and Ohio tomatoes or the pronounced variations shown by these molds in their ability to produce Physical and chemical changes in tomato fruits. It appears that species of Fusarium, 3.422231: Oospora and We may be found more commonly in field tomatoes than 8°n°rally has been noted in the literature. In this study Other molds such as species of Trichoderma, Mycelia sterilia and hormodendrum were found in addition to those which have b°°n associated generally with tomatoes. It is likely that many other genera occasionally may be found on tomatoes and, "fills they may contribute to Howard mold counts, they may be resPonsible for only slight or no damage to tomato fruit. It would appear that in accordance with the many vari- ables associated with the pathogenicity of organisms attacking the human body, there are many factors which influence the par'fisitic behavior of molds affecting tomatoes. It is ap- par-ant that one should not regard all molds as being of equal 8181lificance in producing rot. Two relatively common molds present during the growing “‘Bon on field tomatoes, Alternaria solani and Colletotrichum W frequently were responsible for only minor blemishes 113 in the tomatoes. By contrast, pronounced degradation was ob- served in tomatoes from which species of Mucor, Rhizo us, Oos ora, Fusarium and some other molds were isolated. Sub- stantially similar results were found upon reinoculation of the molds into tomatoes. It was interesting to observe that when Rhizopus giggi: m, the common bread mold was introduced into tomatoes a rupturing of tissue resulted. The cause of cracks in tomatoes has been reportedly obscure (Howard, 1937). It may be that the rate at which molds liberate enzymes into tomato tissue 13 responsible, as well as weather conditions, for some of the era eks which are found in tomatoes. Correlation 01' the StUdy shows that g. nigricans produced a high concentration 01' the enzyme PG in tomato juice and liquefied pectate gel. It appeared representative of molds which behaved in sub- stantially the same manner as species of 34229; and Oospora. By contrast, cultures of other molds, particularly 8pecies and strains of the genera Alternaria and Colletotrichum generally did not cause severe damage to tomato tissue in a Short period of time. These same molds produced little or no d°tectable PG in any of the media tested. It seems that PG profiliced byumost tomato molds is the principal agent by means of which molds damage tomato fruits rapidly. It is contended that it is the relative amount of enzymes produced by these molds, not their presence or absence which is of prime impor- tan(to. This is in agreement with the views eXpressed by Holden (1950b). 11h The instance in which the Howard mold count showed excess of 50 percent positive fields when only 0.1 percent visible rot was present emphasizes how a low percent of visible rot may mean Government seizure of a pack while at other times this amount of visible rot would yield an acceptable Howard mold count. That high mold counts can be produced when such minute amounts of visible rot are present may eXplain why some canners have difficulty in complying with Government mold count standards. This would seem to apply particularly when only minor blemishes such as might be caused by Alter- 2221‘s. and Colletotrichum appear on tomato fruit. One should not ignore the fact that the size of mold filaments also would be eXpected to have an appreciable in- f1Hence on mold counts (Beneke, 1950). This might explain even more fully how high mold counts can be obtained when the amount of visible mold damage is slight. Similarly, the fragility of the mold fragments logically would assume impor- tance. Darling had similar views (1922). It was noted that the amount of viSible mycelium pro- du°°d by a mold was not related to the amount of pectolytic enames produced by the mold. This substantiated the results I"E")°~'l"t.ed elsewhere (White and Fabian, 19533 Fernando, 1937). Mold counts made on tomatoes from which—all signs of ' V18ible rot had been removed were almost consistently higher than counts obtained by comminuting tomatoes which did not re- quire any trimming. This confirms similar findings noted by ”and 335;. (1953). 115 The question arises as to what a processor can do if con- fronted by a poor growing season and the necessity of exten- sive trimming of the tomatoes. It is obvious that he is placed in an unfortunate financial position as a result of ex- tensive labor costs and, in addition, may be confronted with a higher mold count than that of a competitor in another sec- tion of the country where tomatoes are produced freer from blemishes and areas which require trimming. Tomatoes from which rot had been removed were less acid than tomatoes which did not require trimming. Analyses showed ‘ j that the pH value of trims and culls ranged from 1+.S - 5.8 as compared to that of 14.3 of the incoming tomatoes. It is not unlikely that some mold growth present in tomatoes may make them less acid and thereby improve the flavor. At the same time it has been suggested that the more alkaline conditions Which prevail could increase the possibility of flat-sour aDozilage. Some processors have contended that tomatoes may show substantial mold growth and yield a better flavored Juice than tomatoes free from mold. This investigation supports the contention that this could readily occur. A generalized statement that all molds improve flavor w(Mild be erroneous but no more so than one to the effect that all molds are harmful and undesirable. Some of the molds, particularly some strains of Aspergillus, Rhizoctonia, Tricho- 'de\1"m§ and Fusarium produced very obnoxious flavors and odors. 116 Other molds, notably Mucor and Penicillium, after inoculation inn) the juice, imparted a sweet flavor during the first few days. After four days a taste panel preferred tomato juice containing the Penicillium strain to uninoculated juice. The production of a high polygalacturonase potency by the molds which rapidly attacked tomato fruits in contrast to the absence or low PG potency associated with the molds which experimentally caused little damage, suggests that the enzyme polygalacturonase is responsible for much, if not all, of the damage done to tomatoes by some molds. The action of Bhigopus when introduced into tomatoes could certainly be ascribed to such enzymatic activity while the failure of Alternaria and Colletotrichum to behave in a similar manner could be explained by the lack of rapid PG production. PG assumed a similar role in the decomposition of black rasp- berries (White and Fabian, 1953), and in decomposition of strawberries (Beneke 23 al., l9Sh). One cannot overlook the degradative action which the molds might produce when allowed to develop in tomato tissue over a long period of time or under other conditions. These have not been con- sidered in this study. Also, mass inoculation has been known to influence pathogenicity. Additional studies of an extensive nature are required to develop greater information concerning the parasitism of fungi in tomato tissue. wright and Brian (1953) reported that they had isolated from some strains of Alternaria solani a product referred to 117 as alternaric acid. This had antifungal properties and was elm: markedly phytotoxic. Less than 1 p.p.m. on reinjection produced lesions in stems and leaves. However, no correla- tum) was found between the pathogenicity of various strains ofrmolds and their capacity to produce this acid. Oxalic acid produced by molds can not be disregarded (Valleau, 1915; Brown, 191,5; Gibson, 1952). Proteolytic enzymes might be considered for more detailed study (Upod, 1952). The role of cellulase might be profitably investigated in greater detail. However, the low potency of cellulase produced by the molds employed in this study and the lack of relationship between it and the severity of attack supports the contention that it is of considerably less significance than.polyga1acturonase in the breakdown of tomato fruits. 118 SUMMARY The principal genera and species of molds found in Indiana and Ohio tomato fruits were determined. The genera Oos ora, Fusarium, Rhizo us, and Mucor were found to be more commonly associated with tomato defects than had been noted previously. By contrast, Alternaria solani and Colletotrichum ghomoides, two widely distributed molds were found to be asso- ciated with only minor lesions in most instances, particularly in the early stages of attack. In addition to the molds commonly associated with tomatoes the genera Trichoderma, Micelia and Hormodendrum were found. Thirty-three cultures of the principal molds were inocu- lated-experimentally into the tissue of sound whole tomatoes and the degree of attack was noted. Alternaria 293333.: .__c°,lletotrichum phomoicLejs, Trichoderma sp. and Hormodendrum sp. p”(minced relatively little damage to the tomato tissue compared to the action of species of $29.93.: Oospora and Rhizopus. The latter was found to rupture tomato tissue and produce cracks in advance of the point of inoculation. The damage to tissue by the molds bore no relationship to the amount of visible Browth. Mold counts were made of tomatoes showing varying per- centages of visible rot and it was determined that a percen- tage of visible rot as low as 0.1 percent could give a mold ‘ __ 119 count in excess of 50. The mold count associated with an es- tablished percentage of visible rot was markedly influenced by the genera of molds present. Average mold analysis could not be expected to know these molds, yet these molds are responsible for the severity of attack, flavor and odor changes. Itwss emphasized that a low percentage of visible rot may mean Government seizure of pack while at another time this amount of visible rot would yield an acceptable Howard mold count. The presence of the molds in tomatoes generally had the effect of making the tomatoes less acid. It was suggested that the increased alkalinity may improve the flavor of tomato juice yet promote the development of flat-sour spoilage. Gom- minuted tomato fruits containing some genera of molds were pleasant and in some instances showed improved flavor compared to fruits not containing mold. The effect of factory procedures during tomato processing was studied. Trimmed tomatoes showed substantially higher mold counts than tomatoes which showed no blemishes and did not require trimming. This raised the question as to what a pro- cessor can do when soil and weather conditions encourage fungal attack. The answer appears to be for Federal, State and local agencies to encourage sound cultural practices. In the fac- tory, it was suggested that careful inspection of final produce going into the product replace the Howard mold count. 120 A study was made of the production of polygalacturonase and cellulase by 120 lmlds when inoculated experimentally into tomato Juice. The potency of polygalacturonase in wholly and partially decomposed areas trimmed from.field tmnatoes which had been invaded by various molds was deter- mined. It was found that the ability of the molds to rapidly attack tomato tissue was coupled with the ability of these molds to produce polygalacturonase. The molds which experi- mentally produced the greatest amounts of PG were isolates of w, Oospora and Rhizepus. The majority of the molds isolated produced only a small amoiant of cellulase and this appeared to bear no relationship to the extent of decomposition associated with any of the molds. It was emphasized that slight changes in the medium, the age of spores, the genus, the species and the strains of the ‘mold markedly influence the ability of molds to invade or penetrate tomatoes. These and other factors suggest the need for additional studies of an extensive nature. 121 BIBLIOGRAPHY Almendinger, V. V., Dillman, C. A., and Beisel, C. G. 195h. Determination of free galacturonic acid in citrus products. Food Technol., 8, 86-88. American Can Go. 1950. The Howard mold count method as ap- plied to tomato products. 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