This d i s s e r t a t i o n

has b e e n m icrofilm ed
e vcrli'; a" 'T-r;-'jved Mic GO— 5443

M E R E K , Edward L. A S T U D Y O F T H E M U M M Y
D IS EA SE O F T H E C U L T I V A T E D M U S H R O O M ,
A G A R I C U S C A M P E S T R I S L. E X FRIES.

The Pennsylvania State University

Ph.D., 1960
Botany

U n iv e r s ity M icro film s, In c., A n n A rb o r, M ic h ig a n

 ;
The Pennsylvania State University'

The Graduate School

Department of Botany and Plant Pathology

A Study of the Mummy Disease of the Cultivated

Mushroom* Aqaricus eamoestris L0 ex Fries

A thesis in

Botany

by

Eduard L„ Mere!:

Submitted in partial fulfillment

of the requirements
for the degree of

Doctor of Philosophy

June, 1950

Approved:

Associate Professor of Botany and Plant

Pathology^ Thesis Adviser,,

<< <->
/ Hoad'-tfx the Departn^ht of Botany and Plant
Pathology
 TABLE OF CONTENTS

Acknowledgments . . . . . . . . . p . . . .

I. INTRODUCTION

General statement of the problem 0 o o o

Origin and importance of the problem .

Scope of the problem

II. REVIEW OF THE LITERATURE

Introduction

Symptoms of the mummy disease . . . . . . p . . . .

Transmission of the disease . . o o o . o . o o o .

Effects of various factors on the infectivity of

soil and compost . . . . . . . e . . . . . .

the causal agent p . p . . . . . 0 . 0 0 . 0 . 0 0 0

III. GENERAL EXPERIMENTAL GROWING PROCEDURES . . . . . . . a

IV. METHODS AND RESULTS OF CORRELATION STUDY BETWEEN

COMMERCIAL GROWING PRACTICES AND THE OCCURRENCE

OF THE MUMMY DISEASE oc, o 0 o o a c o a o & o ° o ° o

V. METHODS At© RESULTS OF MICROFLORA AND MICR07AUNA

ISOLATIONS

Fungi . . p . . * 0 . 0 0 . 0 0 . o o o . . 0 0 0 0

Nematodes . . p p . . . . 0 0 . 0 0 0 0 0 0 0 0 0 0

Protozoa . p a . p p . o . . . o . o o o o p . . o

VT. METHODS AND RESULTS OF TRANSMISSION EXPERIMENTS

VII. DISCUSSION

Correlation between commercial growing practices and

and the occurrence of the mummy disease c *

 F.Iicroflora and microfauna ,. • .........

The nature and habitat of the causal organism

VIlI, SUMMARY e « a a a « »« a e o o o e o a o e

LITERATURE CITED..... ............... . .* • o . » o „ *

APPENDIX Cooperating mushroom growers o o g , a o „ ot,

 Acknouledgmsnts

The guidance and encouragement of Dr„ Leon £i„ Kneefoone, thesis

Adviser and Director of Ehishrocm Research at The Pennsylvania State

University, is gratefully acknowledged,, The following acknowledgment

are also gratefully made to: Mrs, Perrina Schultz for many mis­

cellaneous aids, the American tlushroom Institute for providing

financial assistance, and Priscilla Uerek for encouragement during

the writing of this thesis,,

 I3 INTRODUCTION

General statement of the problem

The causal agent of the mummy disease of the cultivated mushroom-

Aqaricus campestris L e ex Friesf has not been identified nor have pre­

ventative measures of the disease been determined,, This study was

initiated to identify the causal agent, to find a preventative measure,

and to establish information and techniques to assist in any sub­

sequent investigations of this disease,,

Origin and importance of the problem

Despite the fact that diseases of the cultivated mushroom have

probably been studied to a greater extent than any other phase of

mushroom growing* there are still several disorders not fully eluci­

dated including among them the mummy diseasen Most Pennsylvania

mushroom growers have had or have seen the mummy disease? and,

although it is not generally responsible for large financial losses

to the mushroom industrys its frequent occurrencer its destructive*-

ness, the magnitude of the disorder in certain cases, the mystery of

its origin and nature, and the possibility of future epiphytotics have

made the growers increasingly concerned about the lack of information

on this diseaseB

Scope of the problem

The limits or inclusiveness of this study could not have been

defined at its commencement since appropriate procedures were always

dependent upon the results of previous observations or experiments

contained herein, Eventually, however,. the following three lines of

approach ware established: 1) an attempt to correlate the appearance

of the disease with some cultural procedure or environmental factor-

2) attempts to associate some organism as the causal agent by making

comparisons between the microflora and microfauna of affected and

normal areas of mushroom beds, and 3) attempts to characterize the

nature or habitat of the causal agent by transmission experiments.,

 II. REVIEW OF THE LITERATURE

Introduction

In 1942, Tucker and Routien (0) published the only <eDropre hens ivo

study of the mummy disease,, Two previous publications 16, 7) ware

essentially progress reports„ The disease was first observed in 1935

in a limestone cave at Auxvasse, Missouri, and later found at llarm-'jnn,

Missouri. In correspondence with Dr, A. M 0 Kligman of Philadelphia,

Tucker and Routien discovered that Dr, Klicman had observed the s-smes

condition at Kennett Square, Pennsylvania, end suggested the cor»j»<jn

name "imuaaiy disease". Whether or not the disease existed prior to

1935 is unknown. Subsequently the disease was identified in. Switzev-

land in 1940 (3) and in England in 1731 (2),

S ymptoms of the mummy, disease

Tucker and Routien (8) described diseased sporophoros of the

white variety of the cultivated mushroom as smaller than normal and

always tan or brown in color; the pilous eccentric or tilted c:-sJv;r

f*‘an perpendicular to the stipe; the gi Lis and annultis often pearly

developed; the stipe longer and smaller in diameter than normal. end

frequently curved; tissues of the stipe and pileg?s usually dry,

tough, and spongy, but in some insta&ces wer.tex on the surface than

ur»mal0

A gritty sound or feeling was obtained when the stipe of on

infected sporophore was cut by a sharp knifo. Water-soaked streaks

were frequently found inside the st ip«» and pi leu r., At a later stage,

these streaks would develop into cavities filled with bacteria, A

small region of brovm discoloration was always present at the point

of attachment of the rhizomorphs to the stipe. Dr. J. Sinden,

according to Atkins (1), suggested the presence of interior pitting

at the junction of stipe and pileus as a diagnostic feature.

Rhizomorphs developed more abundantly on diseased sporophores

than on normal sporophores; and excess tissue, frequently forming a

mat. was found at the base of the stipe (8). Both the rhizomorphs.

except for their profuse development, and the mycelium in the soil

and compost appeared normal.

Diseased sporophores appeared initially in a small area of the

bed and subsequently sporophores which appeared in adjacent areas

became diseased. Normal mushrooms were rarely found in affected

areas, and although new pinheads formed, most displayed mummy

symptoms.
Comparison of soil and compost from areas producing normal

sporophores and areas producing diseased sporophores indicated no

differences in color, moisture content, texture, odor, pH. or the

abundance of nematodes, fungi, or insects. Thomas and Mitchell (5)

stated that "mummy” type mushrooms were associated with "large

numbers of nemas in the soil and on the broken ’roots® of these mush­

rooms, and the disease spreads along the bed in the same manner and

at the same rate as do the nemas in the manure."

Transmission of the disease

Isolations and inoculations

Mushroom decoction, potato, potato dextrose, asparagine, tap

water, and nutrient agars were used in attempts to isolate micro­

organisms from the soil, compost, and diseased sporophores (8),

Six different species of bacteria were isolated from the pilei

and stipesa No single species could be associated with the disease,,

The bacteria were incubated in nutrient broth, then poured on the

soil and into the compost of normal mushroom trays and injected into

normal sporophores and buttons but no abnormalities resulted from

these treatments. Similar studies conducted by Fluery (3) confirmed

these results.

Isolations from the basal portion of the stipe yielded species

of Trichodertna, Elucor. Fusarium, Aspergillus, Verticillium, Penicil-

lium. Rhizopus. Cephalosporium., and Pythium (8), None of these

organisms proved pathogenic when grown on agar slants and placed into

compost and soil of producing trays. Isolates from normal and infested

soil were representative of the common soil microflora.

Transmission by casing soil and compost

'‘The only method by which the mummy disease has been transmitted

from diseased to normal beds involved placing casing soil or compost

from a diseased area in contact with a normal bed" (8), Sections of

soil and compost (about two square feet) were cut from an area pro­

ducing diseased sporophores and placed into a normal bed with contact

between the soils and the composts. The disease appeared in sporo­

phores of the normal section in seventeen to twenty-five days follow­

ing the transfer, Later experiments showed that either the soil or

the compost from the area of diseased mushrooms could transmit the

mummy disease x^hen placed into a normal bed. The soil within five
 6,

feet of the symptomatic area also exhibited this infectivity.

Attempts to transmit the disease with affected sporophores or

rhizomorphs gave negative results (8)0 Soil on which diseased

sporophores had appeared was stirred with water and allotved to settle*

The soil as well as filtered and unfiltered leachates were placed

into normally producing beds. The mummy disease appeared only on the

beds treated with the soil.

Effects of various factors on the infectivity of soil and compost

Aging

Soil and compost in affected beds retained their ability to

transmit the disease at least eight months when stored in a humid

cellar and kept moist C8). Infective casing soil placed in barrels and

kept in the cellar for six months lost its infectivity. Soil placed

in a tightly closed container and stored at room temperature for

seven weeks did not transmit the diseasec

Temperature

Containers with 15 liters of soil or compost were wrapped with

heavy paper and stored at -12° C S( 4.5° C oe and cellar temperature

(15-23° C a) for four months (8). The soil and compost stored at

4.5° C. and at cellar temperatures transmitted the disease when

placed into normal beds.

Containers with only one liter of soil were stored for four

months at various temperatures and under different moisture conditions.

None of these samples retained their infectivity.

Heating infective casing soil at 55° C„ for two hours„ steaming

at 100° C. for two hours, or autoclaving for twenty minutes rendered

the soil non-infective, "Suspensions of infected soil in water were

maintained ten minutes at temperatures of 47°* 57°, and 67° C." This

soil treatment also rendered the soil non-infective, No indication

was given of the quantity of soil used.

Chemicals

Twenty milliliters of formaldehyde were mixed with 18 liters

of infective soil, and one ounce of carbon disulphide with 324 cubic

inches of infective soil (8). The carbon disulphide destroyed the

infectivity but the formaldehyde did not.

Five hundred grams of infective soil were treated (8) with the

following chemicals: carbon disulphide, 5 ml.; formaldehyde, 10 ml*;

calcium cyanide, 5 g.j trichlorethylene, 2 ml,; tetrachlorethane,

2 ml.; pentachlorethane, 2 ml.; perchlorethylene 2 ml.; Ceresan (5%)c

2 g,; pyroligneous acid (16 parts to 100 parts water), 40 ml0; ammonium

hydroxide (17.5%), 5 ml.; and acetic acid (1.2%), 8 0 ml. The

infectivity of the soil was destroyed by all these treatments except

the pyroligneous acid and the ammonium hydroxide treatments.

The causal agent

Since Tucker and Routien could not find any causal organism, they

postulated the existence of a virus (8). "The transmission of the

mummy disease to normal beds may be dependent on anastomoses beticeen

normal and parasitized hyphae,"

Bawden and Gregory (2) attempted to test this postulate. Extracts

from mummy mushrooms were added to compost with the spawn and injected
into young buttons. Pure cultures of the Aqaricus campestris

mycelium were obtained from pileus tissue and from spores of diseased

sporophoreso Spawn was made from these isolations and planted in

mushroom beds. The disease was not transmitted by any of these methods,,

and they concluded that “The disease appears not to be caused by a

virus that is present within the hyphae or spores of affected fruiting

bodies."

The source of infection

The source of initial infection is unknown (8). The suscepti­

bility of the agent to heat suggested unsterilized casing soil as a

likely suspect.

Control

The spread of the mummy disease through a bed was controlled by

cutting a six-inch wide trench at least six feet in advance of both

ends of the affected area. Wo other method of control is kncvm (8).

 90

III, GENERAL EXPERIMENTAL GROWING PROCEDURES

All experimental crops were grown in the north room of the old

experimental mushroom house on the campus of The Pennsylvania State

University, University Park, Pennsylvania,, The trays were arranged in

a checker-board fashion on six shelves. The room held approximately

100 wooden trays having inside dimensions of 18 inches in length, 15

inches in width, and 5 inches in depth. Air temperature was the m o -

statically controlled; heat being provided by a hot water system and

cool air by an air-cooling unit employing ammonia as a refrigerant.

The entrance of outside air was regulated by vents in the false

ceiling.

Compost was made from fresh horse manure obtained from the

riding stable on the campus. Approximately one and one-half tons of

manure, enough to fill the trays for the growing room, were formed

into a pile four feet wide and three feet high with the aid of a

small custom-built compost turner. Turnings followed on the second

and fifth days, or second, fourth, and seventh days with the filling

of the trays on the eighth or ninth day, respectively. Gypsum and a

nitrogenous supplement, either brewers’ grains or poultry manure, we re

added to the pile on the second turn. The trays were hand filled with

the compost and stacked in an improvised pasteurizing chamber fitted

with steam pipes. The trays of the compost were pasteurized at 130-

140° F, for four days and then allowed to cool.

Rye grain spawn of The Pennsylvania State University culture 286

was sown on top of the compost of the cooled trays and they were then
arranged in the growing room, After two to three weeks of incubation

at 70° F„c the trays ware cased with one inch of steam-treated top soil

from The University farms and the room temperature reduced gradually

until it reached 60° F* as the mushrooms began to appear. Mushrooms

were examined,, counted, picked and weighed almost every day for forty

to £lxty days (cropping period)? Due to the proximity of the hot

water pipes, the casing soil dried rapidly and had to be watered

almost daily during cold weather. No diseases became troublesum *

Ousting with zineb eliminated or checked the few Vertieillium

infections which started, Phorid flies were frequently annoying and

could not be controlled by DOT or methoxychlor, but were almost

eliminated in the spring of 1957 by painting the woodwork with

uiazinon.

At the end of a cropping period, the trays were removed from

the growing room, carefully inspected, and the compost discarded

The growing room was cleaned out thoroughly and the woodwork weLluu

with Elgetol or Terrachlor (PCNB) solution. All trays had been

treated with a copper naphthenate (Rot-uot) solution prior to their

use in these experiments.

The results of previous crops grown in this manner indicated

a production of 100 to 300 mushrooms and a cut weight of 2 to 4

pounds of mushrooms per tray. Yields within these limits have been

considered as normal production for the purposes of this study.

 IV. METHODS AND RESULTS OF CORRELATION STUDY BETWEEN

COMMERCIAL GROWING PRACTICES AND THE

OCCURRENCE OF THE MUMMY DISEASE

Methods

On July 29, 1955, a form letter was sent to thirty mushroom

growers and also printed in the A, M. I, News, the monthly publi­

cation of the American Mush room Institute, Kenriett Square, Pennsyl-

vania, The content of the letter was as follows: "With the aid of

the A. H. I, grant, I have just started to work on the mummy disease

under Dr, Leon R. Kneebone at The Pennsylvania State University, In

order to follow the progress and occurrence of the disease, and to

obtain diseased material for experimental work, I intend to keep

records on all mushrcos houses which shew signs of the disease. I

would greatly appreciate your sending me a card if you or any other

grower in your vicinity suspect mummy at any time."

Almost all growers who reported a case of the mummy disease tvere

visited, A few could not be visited because of the amount of time

involved in traveling. Diseased areas we re inspected for the pre­

sence of organisms and growers were questioned on the following points

of interest;

I, General sanitation

2, Compost materials

3, Pasteurization

4C Origin of spawn

5. Soil treatment

6, Management of crop
 7, Time of appearance of disease

8* Progress of disease

Results

Between June, 1955 and April, 1956, fourteen occurrences of the

mummy disease were examined at commercial growing plants* Several

other plants were visited in response to calls but are not included

in this study because the diagnosis of the abnormal condition was

inconclusive. The names and addresses are listed in the Appendix,

General sanitation. The general sanitation in and around the

plants of nine of the fourteen growers was excellent. Although only

one visit was made to most of the plants, these nine growers appeared

to know and practice the recommended precautions. The other five

growers were lax in various ways such as composting on a dirt wharfc

piling trash outside of the houses, dumping compost adjacent to the

growing houses, and providing improper drainage of the grounds around

the plant. Because of precautions taken by the nine growers, however,

it was impossible to conclude that any deficiency was common to most

or all of the growers visited.

Compost materials. Eight growers used synthetic compost the bulk

ingredients of which consisted of hay and corn cobs, five growers used

horse manure, and one used a mixture of synthetic compost and horse

manure. Hie number of growers using synthetic compost appeared to be

out of proportion to the number of such growers in Pennsylvania, but

again no conclusions could be drawn from this fact alone. Three of

the growers using synthetic compost were .located ,at £3t„ Pocono,

Pennsylvania, where one grower sent a report whenever anyone had

troubles* and five growers using synthetic compost were located at

Temple* Pennsylvanias where one progressive groiwer sent notification

whenever he heard that someone had mummy* Most of the growers at Mt*

Pocono used synthetic compost* and probably about ninety per cent of

the growers in the Temple area used synthetic compost,. It seemed

likely that the high proportion of growers using synthetic compost

was due to the energetic character of two growers who lived in these

areaso There is no way of knowing whether the growers using manure in

Chester County had very little of the- mummy disease or whether they did

not report it„

Pasteurization,, Most of the growers visited did not keep any

records of their pasteurization process but seemed to think or remember

that the temperatures were between 130° F„ and 140° F„ * the recom­

mended temperatures0 Three growers reached peak temperatures of 148°

F<, * 155° F.„ and 156° F„ Too many variables, such as memory of the

grower9 temperature differentials between beds* and duration of peak

heat* made this information useless in analysis,,

Origin of spawn„ Spawn from ten different commercial spavm

makers was available to the growers of Pennsylvania at the time of

this study and the mummy disease was found to appear in the mushrooms

produced from nine of these ten sources,, Mummy occurrences at the

Butler County Mushroom Farm* which has its own unique culture*

appeared in trays at random,, although "stroma"* a distinct type of

abnormal mycelial growth which was definitely associated with spawn„

appeared in four adjacent trays which had been spawned from the same

one-gallon jug of spawn,,

 14.

Soil treatment. Four growers treated the casing soil with steam,

seven with steam and formaldehyde, and four used no treatment at alle

It appeared that soil treatment has no influence on the occurrence of

the mummy disease, but such conclusions are not justified. Efficient

soil treatment is influenced by many factors, such as time and thorough­

ness of treatment, moisture content of soil, and soil handling after

treatment. Most of the growers could not express these factors in

quantitative terms or were not aware of them. It is possible that

all of these growers who treated their soil did so inefficiently, so

no conclusions can be drawn with regard to the disease.

Management of the crop. The purpose of this phase was to deter­

mine if any particular watering practices seemed to be correlated with

the appearance of the mummy disease. At the beginning of this study,

several growers had expressed opinions that mummy occurs more fre­

quently following heavy waterings. The attempt to obtain any useful

information on this point from the growers who were visited, however,

was a failure because quantitative data could not be obtained.

Time of appearance of disease. All but one grower first noticed

symptoms of the mummy disease on the first, second, or third flushes.

The first symptoms seemed to appear in equal numbers on all three of

these flushes. One grower noticed it only on the fifth break. There

was no correlation with the time of appearance and the time of the

year.

Progress of the disease. The growers' observations indicated

that the mummy disease spread in all directions at the approximate

rate of one foot a day unless it was stopped by a trench cut at least
 1n :

six feet in advance of the peripheral symptoms* The only except-i».n

to this was one case in which the disease progressed in ^ .>•.'

in one direction but made no progress at all in the other direction;

No areas ever produced normal mushrooms once the typical symptoms oi

mummy had developed in the area*

 16.

V« METHODS AND RESULTS OF MICROFLORA AND

MICROFAUNA ISOLATIONS

Fungi

Attempts were made throughout this study to associate some

particular fungus or fungi with the occurrence of the mummy disease.

All isolations ware made in 90 millimeter Petri plates containing

approximately 20 ml- of agar* Each, plate a'ivrays contained four

different. deposits of material from which the isolation was attempted.

From compost samples, small pieces of straw were placed on the plates;

from soil, small particles of soil; from rhizomorphse approximately

1 «r<m„ of a strand; from sporophore tissue, small pieces of tissue

removed aseptically from inside the sporophore. All plates were

stored at room temperature for at least two weeks before discarding.

All media contained 17 g* of Difco agar per liter and were auto-

;iaved at 121° C 3 for 15 minutes. The following types of media were

used;

Malt Agars

Fifteen grams of Difco Halt Extract (1136)

per liter.

Cornmeal A qar;

Seventeen grams of Difco Cornmeal Agar CB386)

per liter.

Nutrient Agar;

Eight grams of Difco Nutrient Broth,

per liter.
 Thirty-five grams of Difco Czapefc Dcx Broth

(B33B) per liter,

Lima Bean Agar:

Twenty-three grams of Difco Lima Bean Agar

(117) per liter.

Potato-Dextrose-Yeast Anar:

Decoction from 250 grams sliced but unpeeled

potatoesg ten grans of dextrose, and lc5 grams

yeast extract per Liter.

Mushroom Decoction Agar:

Decoction from 250 grams of washed mushrooms

per liter.

All isolations or observa 11 ans of microorganisms will be

tentified as "Observations”.

Observation

Methods. Isolations ;ere made from soil and compost from an

re a of typically mummied mushrooms from Breezyacres Mushroom Co* Temple,

rnnsylvania, collected June, ID55, Three plates of each material

ere made on malt agar and potato-dextrose-yeast agar.

Results. Trichoderma viride was isolated on malt and

potato-dextrose-yeast agars from samples of soil and compost from

oth affected and normally producing areas.

Observation 2.

Methods. Isolations ware made from soil and compost from

r.rmal areas and soil and compos I from affected areas from Butler
 180

County Mushroom Farm, West Winfield, Pennsylvania, August, 1955, Five

plates were made of each material on malt agar and potato-dextrose-

yeast agar.

Results, Trichoderma viride was isolated on malt and

potato-dextrose-yeast agars from samples of soil and compost from both

affected and normally producing areas. This organism could not be

observed on visual or microscopic examination of the soil or compost

samples and may have been growing in the soil and compost without

sporulating or not growing there at all. Perhaps spores from these

materials germinated on the agar.

Observation 3.

Methods, Isolations were made from soil and compost from

affected and normal areas from the mushroom plant of John Caliguiri,

Mt, Pocono, Pennsylvania, collected in February, 1956, Five plates

were made of each material on cornmealt nutrient, malt, and mushroom

decoction agars.
 19.

Results: The results obtained are listed in Table 1,

Table 1„ Fungi isolated during Observation 3„

Materials Media No* of Time 3 Organism

Isolated

Normal soil Cornmeai agar 20 Trichoderma viride

Malt agar 20 Trichoderma viride
Nutrient agar 4 Mucor sp.
16 Trichoderma viride
Mushroom agar 20 Trichoderma viride

:!u(i0ny soil Cornmeai agar 20 Trichoderma viride

Malt agar 20 Trichoderma viride
Nutrient agar 4 Bacillus mycoides
14 Trichoderma viride
2 Fusarium sp,
Mushroom agar 16 Trichoderma viride
4 Mucor sp,

Normal compost Cornmeai agar 20 Trichoderma viride

Malt agar 20 Trichoderma viride
Nutrient agar 20 Trichoderma viride
Mushroom agar 20 Trichoderma viride

Mummy compost Cornmeai agar 20 Trichoderma viride

Plait agar 20 Trichoderma viride
Nutrient agar 20 Trichoderma viride
Mushroom agar 20 Yellow Trichoderma
viride

Organisms which may have 'hod some relation to the disease were

-obtained from mummy compost or mummy, soil and not from normal soil or

ooapost# Infestation trials were made with two organisms, the yellow

Trichoderma viride and the Fusarium sp„ f in Experiment ?>a

Observation 4.

Methods* Isolations were made from soil and compost, and

f?:oivorphs from affected and normal areas from the mushroom plant oi
 20.

William Ghione, Kennett Square, Pennsylvania, collected in May, 1956.

Three plates were made of each material on malt, Czapek Dox, potato-

dextrose-yeast, and lima bean agars.

Results. Two hundred and eighty-four samples from normal

soil, compost and rhizomorphs and from soil, compost and rhizomorphs

taken from an affected area resulted in the isolation of Trichoderma

viride in malt, Czapek Dox, potato-dextrose-yeast, and lima bean

agars. An Acremoniuro sp. was isolated from four different rhizo-

morphs taken from an affected area. This organism was tested for its

ability to induce the mummy disease in Experiment 3.

Observation 5.

Methods. Isolations were made from soil and compost from

normal and mummy areas and areas producing ”open-»veil" mushrooms from

The Pennsylvania State University, November, 1956. "Open-veil" mush­

rooms are sporophores with hard undeveloped gills. Each material was

placed into holes cut with a #6 cork borer into three apples and three

potatoes. The holes were sealed with Scotch tape and left standing

at room temperature for one week. The apples and potatoes were then

cut open and four pieces of tissue removed from the margin of each

hole and placed on potato-dextrose-yeast agar plates.

 Results& Tiie results are given in Tabic 2

Table 2 ? Incidence o±‘ fungi isolated during Observation 5

Potato

Organism Normal Open-veil Mummy

soil compost soil compost soil compo

Penicillium spa 1 I
Phycomycete 3
Stysanus 8 3 i
Fusarium spK 3
Dictyostelium sp. 12

Apple

Organism Normal Open-yeil Mammy

soil compost soil compost soil compo

Penicillium spn 8

Phycomycete
Trichoderma spe 12 i2 12 2 10
Chaetomium sp„ 2
Alternaria sp, 3
Dictyostelium sp; 3

The unidentified phycomycete,-, the Fusarium spi;. ant! the Oicty

stelium sp, were placed into healthy mushroom trays in Experiment

The Alternaria spa was not tested because it was apparently asroci

with a soft spot in one of the apples previous to the inoov. 1 it ion -

Observation 6 ^

Methods o Each tray from Experiments 5 and 6 was exe mi ne­

at dumping time. The condition of the spawn and the presence o:i
foreign organisms were recorded for each tray*

Results, The results are given in Table 3.

Table 3, Number of mushroom producing trays exhibiting

abnormal characteristics

Abnormal Characteristic Mushroom Production on Tray

normal open-veil mummy

None 22 3 3
yellow mold 6 7 0

Trichoderma sp 6 18 6 0

wet compost 5 6 0

sparse spawn 4 2 1

no spawn 1 1 1

Chaetomium sp„ 6 5 1

Stysanus sp„ 3 I 0

wet compost and sparse spawn 3 1 0

yellow mold and Chaetomium 2 2 0

yellow mold and Stysanus 1 1 0

Trichoderma and Stysanus 3 3 0

yellow mold and Trichoderma 3 3 4

Stysanus and Chaetomium 0 3 0

Trichoderma and Chaetomium 1 0 0

Trichoderma and Stysanus and 0 1 1

yellow mold

None of the observed organisms or conditions could be specifically

associated with trays bearing mammy or open-veiled mushrooms,,

Observation 7.

Methods. Isolations were made from soil and compost from

normal and affected areas from the mushroom plant of Ettore Camilli 6 -

Sons, Temple* Pennsylvania, collected June 23, 1957, and subjected to

fiiermal treatments* Three plates were made from each treatment*

 Results, Tables 4a and 4b summarize the results of iso­

lations made from materials which had been subjected to thermal treat­

ments* Each of the various bacteria isolated had distinct colonial

characteristics which made their separation possibleQ These bacteria

were not identified but were maintained in pure culture until the

results of the concurrent Experiment 13 were available. If any of

these bacteria would then have been found to be associated with the

transfer of the mummy disease, intensive studies would have been made

on the pertinent organisms*

 Table 4a* Incidence of organisms isolated from soil during Observation 7,

Treated Mummy Soil

Organisms Untreated Untreated 400 C, 700 c. 100° C,
Normal Soil Mummy Soil 15 30 45 60 15 30 45 60 15 30 45 60

Trichoderma sp. 20 18 12 7 9 7
Mucor sp,. 17 3 4 4 3
Dictyostelium sp. 2 6 1 4
Penicillium sp. 1 1
bacterium #1 6 12
bacterium ^2 20 16 1
bacterium ^3 12 11 12 12 12 12 9
bacterium #4 4 1 10 7 1
Time in minutes)
Table 4b. Incidence of organisms isolated from compost during Observation 7,p

Treated Mummy Compost

Organisms Untreated Untreated 40° C, 70° C, 100° c„
Normal Compost Mummy Compost 15 30 45 60 15 30 45 60 15 30 45 60'

Mu cor spa 6
Trichoderma viride 2 1 2 8 8 12
Dictyostelium sp. 4 1
Penicillium sp„ 1 4
Agaricus csmpestris 8 2
Stysanus sp. 3 1
bacterium W 1 5 10 12 12 12 12 12 12
bacterium ff2 14 14
bacterium *3 12 12 12 12 11 1 1
bacterium ^4 1
bacterium p5 4
iatodes

Nematodes were collected from soil and compost by placing 25 grams

moist soil (as collected) or 50 grams of moist compost {as collected)

o a folded, six inch square of muslin and placed into the top of a

iss funnel* The 1ower end of the funnel was covered with a rubber

e to which a closed metal clamp had been attached and the funnel

- filled I'Jith water* After twenty-four hours, the clamp was re­

used and the water drained into a collecting vessel *

In some initial work, an estimate of the nematode population was

.ained by dropping approximately one milliliter of water from the

^ of the rubber tube into a Syracuse dish and recording the number

nematodes on the dish* The following recording scheme was used to

iipare the average number of nematodes per low power field of the

;roscope:

Table 5* Comparisons of numbers of nematodes

No* of Nematodes Recorded as

0 0

1-3 r

4-9

10-20

over 20

Ln later studies, however, dilutions were made from measured

l'.ber of nematodes per gram of wet soil or compost

 26.

Observation 8 .

Methods. Population estimates of nematodes were made from

affected and unaffected trays of Experiment 1. Materials were ob­

tained from affected trays five weeks after appearance of the diseasea

Ten samples of soil were taken from affected trays and eleven samples

of soil were taken from normal trays. No compost samples were taken.

Results. One sample from the normal soil contained 1 to 3

(jO nematodes. All other samples from normal and mummy soil con­

tained more than 20 (^/AO nematodes.

Observation 9.

Methods. Population estimates of nematodes were made from

soil and compost samples from the mushroom plant of Frank Evanno,

Temple, Pennsylvania, October, 1955. These samples were obtained

approximately ten days after the appearance of mummy symptoms. Three

replicates of soil and compost were taken from the center of the

affected area, from the edge of the affected area, and from outside

the affected area.

Results,, Two samples of mummy soil taken from the center

of the affected area contained 1 to 3 (jO nematodes. All other

samples from normal soil and compost and from mummy soil and compost

contained no nematodes at all.

Observation 10.

Methods. Population estimates of nematodes were made from

soil and compost samples from the mushroom plant of Raymond Gaspari,

Temple, Pennsylvania, October, 1955. These samples were obtained

approximately ten days after the appearance of the mummy symptoms.

 27,

Three replicates of soil and compost were taken from the center of Luo

affected area and from three feet beyond the affected area.

Results, The results are given in Table 6 ,

Table 6 , Estimated populations of nematodes

Normal Mummy
soil compost soil compost

/ // / /
0 0 0 /
O 0 0 /

Observation 11.

Methods, Population estimates of nematodes were mad a

soil and compost samples from the growing plant of Brandywine M .7-

room Corporation, West Grove, Pennsylvania, Novembere 1955e

samples were obtained approximately three weeks after the appearan-V;

of the mummy symptoms. Four replicates of soil and compost wove

taken from the center of the affected bed and from a bed pvoauoiu*;-

normally.

Results. No nematodes were found in any samples ox sotI

or compost from affected or normal areas.

Observation 12.

Methods. Population estimates of nematodes were rued, * :

soil and compost samples from the mushroom plant of John caliQui :*i ,
■

Mt, Pocono, Pennsylvania, February, 1956, The samples were o b U M d

approximately six days after the appearance of the mummy sympi

Four samples of soil and compost were taken from a normal avoa
 28.

and from the center of the affected area.

Results., The results are given in Table 7.

Table 7. Estimated populations of nematodes

Normal Mummy
soil compost soil compost

U m 0

H U •f HU 0

+ u u •f
u u u u it

Observation 13.

Methods. Population estimates of nematodes were made from

soil and compost samples from the mushroom plant of William Saganich,

Mte Pocono, Pennsylvania, February, 1956. These samples were obtained

approximately two weeks after the appearance of the mummy symptoms.

Three replicates of soil and compost were taken from the center of

the affected area and from outside the affected area.

Results. All samples from both normal and affected areas

contained more than 20 iUW) nematodes.

Observation 14.

Methods. Population estimates of nematodes were made from

soil and compost samples from the growing plant of Penn Mushroom

Company, Temple, Pennsylvania, February, 1956. These samples were ob­

tained approximately ten days after the appearance of the mummy

symptoms. Four replicates tr-re taken from soil and compost within the

affected area and from a normal area beyond the affected area.
 29,

Results,. The results ait ryl-ven in Table 8 »

Table 8 , Estimated pop:;'it irons of nematodes

Normal Mummy
soil compost il compost

"it.xon 15.

Methods, Population env- ,. of nematodes were taken at the

of Experiments 5 and 6, .eree replicates were taken from

orrpost of normally pro dr.- .• ;•:? trays ff from trays which rad

ten-veiled mushrooms, and i m trays which had produced,

sbrooms* Seven samples ^ ..re taken from soil and compost

•■vo beds which had produ^-v:* mummied mushrooms*

jiesnits. The results ar- ,;pvan in Table 9,

 30*

Table 9. Population count of nematodes at the end of

Experiments 5 and 6 in numbers/gram wet weight

Soil Compost

Normal trays 0 0

0 0

0 0

Open-veiled trays 0 0

0 0

0 0

Mummy trays 26 0

25 0

25 0

Mummy beds 40 150

31 91
64 208
18 100

30 142

From Table 9 it appears that the presence of nematodes in the

soil may have been associated with the cmmay disease itself or merely

placed into the trays and beds during the infestation procedure and

appeared incidental to the appearance of the disease* -4n infestation

experiment was later made with these nematodes (Experiment 13)•

Protozoa

Observation 16,

Methods, Soil and compost from normal traysp trays with

open— veiled mushrooms* and trays with mummied mushrooms were examined

during the course of Experiment 6 , Small samples of compost and soil

were examined microscopically after making suitable suspensions from

fresh soil and compost. Stains were applied in attempts to discover

 31,

any protozoa which would be difficult to see otherwise, No special

isolation or enrichment techniques were used in this, procedure,

i
Results, Two types of protozoa were found<in all soils„

either fresh or soaIcede and none in any compost samples,, The

Paramecium sp, and a small flagellated form resembling a rotifer

seemed to be abundantly distributed throughout the soils from normale

open-veil producinga and mummy producing trays. No other protozoa

could be found.
 320

VI 0 METHODS AND RESULTS OF TRANSMISSION EXPERIMENTS

All infection experiments but one were conducted in the eld

experimental growing house on the campus of The Pennsylvania State

University, University Park, Pennsylvania,

Experiment 1,

Methods, The purposes of this experiment were to determine

whether;

a, the disease could be transmitted to mushrooms

grown in trays in the experimental house at The

Pennsylvania State University,

b, potentially infectious soil, compost, or both

would be required to transmit the disease,

c, the success of the transmission would be

dependent upon the stage of the affected area

as determined by the symptoms on the bed.

Materials for this experiment were collected from the mushroom

plant of Armando Uorganti, Temple, Pennsylvania, in September, 1955,

Sections two square feet in size were removed from the center of an

affected area, from the edge of an affected area, and from one foot

beyond the affected area. Portions of these sections were placed

into newly cased trays in the experimental house the following day.

Compost infestations were made by removing a cube of compost of

approximately four inches and replacing it with a similar size sample

of compost from an affected area. Soil infestations were made in the

same manner using approximately 16 cubic inches of soil from an

 33c

affected area, Soil and compost infestations viere made by com­

bining both treatments in the same area of the tray, Thus nine

different treatments resulted from the combinations of three

different areas of the bed with soil,, with compost, or with soil and

compost„ Each treatment was replicated twice, The trays were

cropped for seven weeks before discarding.

Results, The results are given in Table 10,

Table 100 Results of treating trays with soil

and compost from diseased areas

Source of materials Materials Replication Results

Center of symptomatic soil 1 np

area 2 np

compost 1 np
2 np

soil and 1 mannay

compost 2 mummy

Edge of Symptomatic soil 1 np

area 2 mummy

compost 1 np
2 np

soil and 1 mummy

compost “ 2 mammy

One foot beyond soil 1 np

symptomatic area 2 np

compost 1 np
2 np

soil and 1 mammy

compost 2 mu may

- normal production
 34#

This experiment indicated for the particular material on hand

that a) the mummy disease could be transmitted to trays in the

experimental hcusev b) the transfer of soil alone (but not compost

alone) might transmit the disease but it was more certain to be

transmitted if soil and compost were used 0 and c) the disease could

be transmitted by using soil and compost from non-symptcmatic areas

at least one foot away from the areas showing symptoms,,

Experiment 2 #

Methods, The purpose of this experiment was to determine

whether or not water infusions obtained from soil and from compost

from symptomatic areas could produce mammy abnormalities,, Soil and

compost from symptomatic areas were obtained from the mushroom plant

of Brandywine Mushroom Corporation, West Grove, Pennsylvania, in

November, 1955, Approximately one pint of soil and compost were

removed from the center of an affected bed, transported to The Univer­

sity in one quart plastic bags, and checked for nematodes# Hie

infusions resulting from these nematode checks were used in this

experiment# Fifteen flower pots having a diameter of ten inches

were filled with spawned compost obtained from the mushroom plant

of Chef Boy-Ar-Dee, Hilton, Pennsylvania# These pots were placed on

the bed-boards in the experimental growing room# The treatments were

as follows:

a, fifty milliliters of tap water poured on

the soil and into the compost#

b„ approximately 150 cc. of casing soil replaced

by an equal amount of soil from an affected area#

 35.

c. approximately 150 cc* of compost replaced

by an equal amount of compost from an

affected area.

d. fifty milliliters of soil infusion poured

on the soil and into the compost

e. fifty milliliters of compost infusion poured

on the soil and into the compost.

Each treatment was replicated three times and the pots were cropped

for seven weeks.

Results. The results are given in Table 11.

Table 11. Results of treating trays with soilf compost 3

and water infusions from symptomatic areas

Treatment Replication Results

Mummy soil 1 mummy

2 mummy
3 mummy

Mummy compost 1 mummy

2 mummy
3 mummy

Control, distilled water 1 np

2 np
3 np

Mummy soil infusion 1 np

2 np
3 np

Mummy compost infusion 1 np

2 np
3 np

The results of this experiment indicated that with the particular

materials on hand the soil and compost infusions made in this manner

could not cause the production of abnormal mushrooms under the experi­

mental growing conditions * Compost alone from an affected area also

transmitted the disease in this experiment which was in contrast to

results obtained with different material in the previous experiment,.

Experiment 3.

Methods, The purposes of this experiment were to determine

whether:
a„ the mummy disease or any abnormalities could be pro­

duced by three different fungi isolated from soil or

compost from affected areas but not isolated from

normal areas ,
b. the mummy disease could be transmitted by potentially

infectious compost which had gone through a maceration

process,:

Ten inch flower pots were filled with spawned compost from the

Chef Boy-Ar-Dee mushroom plant, cased, and placed on the floor of the

basement of the greenhouse in Buckhout Laboratory, The Pennsylvania

State University, IV/o slants of potato-dextrose-yeast agar over­

grown with the fungi to be tested were placed into each flower pot

so that the slants ware in contact with both the compost and the

casing soil. Of the three fungi tested, two were obtained in

Observation 3 (the yellow Trichoderma sp. and the Fusarium sp.) and

the third in Observation 4 (Acreraoniuca sp,),

A suspension of compost from which mummied sporophores had

appeared at the Butler County Mushroom Farm, VJest VJinfield;. Penn­

sylvania, was poured into the compost of three pots. This suspension
 37.

was made by macerating 150 ml* of compost in 150 ml, of water in a

Waring Blendor.

Three pots were treated with soil and compost from an affected

area from the Butler County Farm and used as controls*

These pots were cropped for six weeks during September and October*

1956.

Results. None of the three fungi nor the control infesta­

tions induced the production of any abnormalities. The failure of

this compost and soil from an affected area to induce the mummy disease

might be explained by the results of Experiment 10 which indicated

that the disease could not be transmitted from the D-26 strain to a

white strain of mushroom.

Experiment 4.

Methods, The purpose of this experiment was to determine

if the mummy disease progresses down a mushroom bed or if the apparent

movement may be due to progressive appearance of symptoms from areas

already infested.

The two lowest beds in the experimental house* each approximately

15 feet long and two feet wide* were filled with compost which had

been pasteurized in trays. One week after casing* a four inch cube

of soil and compost was removed from the end of each bed and replaced

with a similar volume of soil and compost from an affected bed in a

mushroom house of John Caliguiri, KJt, Pocono* Pennsylvania. These

beds were cropped for fifty days,.

Results. The results are given in Table 12,

 38.

Table 12* First appearance of the mummy disease

in days after first break.

Replication Distance from point of infestation (feet)

0 3 9 12 15

1 2 9 30 30 40

2 1 4 15 24 42

Table 12 indicates that the symptoms progressed a little less

than one-half foot a day. This was considered a good agreement

with growers? observation that the disease spread approximately one

foot a day.

Experiment ji.

Methods. The purpose of this experiment was to determine

if soil and compost from an affected area would retain their ability

to transmit the disease after being exposed to the vapors of propy­

lene oxide.

The source of the mummy soil and mummy compost was identical to

that in Experiment 4. One liter of the soil or the compost was

exposed to 6 ml. of propylene oxide in a ten inch desiccator for 43

hours. This soil or compost was then used to replace the soil or

compost removed from newly cased trays. The treatments and number of

replications were: control, no treatment (six); mummy soil (two);

mummy soil exposed to propylene oxide (four) ; mummy compost (two);

mummy compost exposed to propylene oxide (four). The trays were

cropped for fifty days.

 39„

Results. The results are given in Table 13

Table 13 . Effect of propylene oxide on infectivity

of rauuiay soil and compost

Treatments Replications Results

Control trays, no treatment 1-6 np

Untreated mummy compost 1 macsiy

2 mummy

Untreated mummy soil 1 mummy

2 mammy

Treated mummy soil 1 np

2 np
3 np
4 np

Treated mummy compost 1 np

2 np
3 np 3 ov®
4 np 2 ov

- open-veil* The number indicates the number of open-veil

mushrooms picfced from the tray*

Experiment 6 .

Methods. The purposes of this experiment isere to determine

whether the water infusions obtained from affected soil and compost

or the leached materials themselves mould induce any abnormalities*

The source of the soil and compost from an affected area was

identical to that of Experiment 4* Four hundred grams of compost

were leached with 1*200 ml* of tap water for two days and 1*000 grams

of soil ware leached with one liter of tap water for two days* Cased
trays were treated with 100 ml. of these infusions or 150 cc. of the

leached soil or compost. The treatments and the number of replica­

tions were as follows: control, no treatment (six); untreated mummy

compost (two); untreated mummy siil (two) 5 leached mummy compost, (two);

leached mummy soil (two); infusion from mummy compost (four); and

infusion from mummy soil (four).

Results. The results are given in Table 14.

Table 14. Effect of leaching on the infectivity

of mummy soil and compost

Treatment Replication Results

Control trays, no treatment 1-6 np

Untreated mummy compost 1 mansny

2 mammy

Untreated mummy soil 1 maimay

2 mammy

Kummy compost infusion 1 np

2 np 2 ov
3 np 10 ov
4 np 9 ov

ESunsny soil infusion 1 np 30 ov

2 np 2 0 ov
3 np 40 ov
4 np 7 ov

Leached mummy compost I mammy

2 mammy

Leached mummy soil 1 mammy

2 mammy
 41*

The results of this experiment indicated that something may be

active in the infusions from the soil and compost from affected ateas

to induce the production of open-veiled mushrooms* Further experi­

ments p.'ere indicated since the controls did not receive infusions

from soil or compost from an unaffected area* This experiment also

indicated that the causal agent was not transmitted by the infusion*

and that leaching did not affect the infectivity of affected compost

and soil*

Experiment 7*

Methods* The purpose of this experiment was to confirm or

reject results of Experiment 6 and determine if recombinations and

different combinations of prater infusions obtained from soil or compost

from an affected area and the leached materials therefrom would

produce any abnormalities when placed in contact with a normal pro­

ducing area*

The source of the mammy soil and compost was identical to that

of Experiment 4 and the materials were leached in the same way as in

Experiment 6 * Thirty-three spawned and cased Azalea pots (fifteen

inches in diameter) were placed on a shelf in the experimental house

and treated with two cubic inches of soil or four cubic inches of

compost; infusions were added in the quantity of 50 ml* per pot*

Each treatment was replicated three times*

Basalts*, The results are given in Table 15*

 42 a
Table 15, Effect of leaching on the infectivity
of mummy soil ati^ mummy compost

Treatments Replication Results

Healthy controls
normal soil infusion 1 np
2 np
3 np

normal compost infusion 1 np

2 np
3 np

leached soil and leached 1 np 1 ov

compost 2 np 2 2 ov

3 np 2 ov

no treatment 1 np
2 np
3 np

Mammy controls
mummy compost 1 tmimasy
2 nansay
3 mammy

mummy soil 1 mummy

2 mummy
3 mummy

mammy soil and mummy compost 1 mummy

2 muEsay
3 mummy

Mummy soil infusion 1 np

2 np 13 ov
3 np 3 ov

Leached mummy soil 1 np

2 np 14 ov
3 np

Mummy compost infusion 1 np

2 np
3 np

Leached mummy compost 1 np 2 ov

2 np 3 ov
3 np
 43.

Experiment 8 .

Methods. The purpose of this experiment mas to determine

if the open-veil condition obtained in Experiment 6 could be trans­

mitted by soil or compost as could the mummy disease or by infusions

of leached materials.

Soil and compost from the trays of Experiment 6 mere preserved

by storing in fifteen gallon galvanized cans in a cold storage room

(50° F.) for one month before being used in this experiment.

Spatmed and cased trays were treated with infusions and leached

materials exactly as in Experiment 6 .

Results. The results are given in Table 16.


Table 16„ Effect of transferring open-veil materials
to normal trays
Treatments Replication
Controls
No treatment
Normal soil infusion
Normal compost infusion
Normal soil infusion and
normal compost infusion
Single Treatments
Ov soil
Ov soil infusion
Ov compost
Ov compost infusion
Combination Treatments
Ov soil and ov compost
Ov soil infusion and ov
compost infusion
Ov soil and ov compost
infusion
Ov soil infusion and ov
compost
44.
Results
1 np
2 np
3 np
1 np
2 np
3 np
1 np 18 ov
2 np
3 np 2 0 ov
1 np
2 np
3 np
1 np 13 ov
2 np 13 ov
3 np 36 ov
1 np
2 np 5 ov
3 np
1 np 12 ov
2 np 28 ov
3 np 2 ov
1 np
2 np
3 np
1 np 6 ov
2 np 6 ov
3 np 10 ov
1 np 7 ov
2 np 3 ov
3 np 10 ov
1 np 1 ov
2 np 7 ov
3 np 10 ov
1 np 5 ov
2 np
3 np 1 ov
 45.

No evidence tsas obtained from this experiment to indicate that

the open-veil condition can be transmitted to a normal tray. Open-

veiled mushrooms appeared predominantly on treated trays but two

control trays also cropped a large number of open-veiled mushrooms.

In any event relatively few open-veiled mushrooms ware harvested from

any treatment when compared to the number of normal mushrooms

harvested.

Experiment 9.

Methods. The purpose of this experiment was to determine

the effect of sterilization on infusions from mummy soil and mummy

compost in regard to their ability to induce open-veil.

Infusions made as in Experiment 8 were antoclaved at 121° C.

for fifteen minutes or passed through a Seitz bacterial filter.

Spawned and cased trays were treated with these infusions as in

Experiment 6 . The used pads from Seitz filters were also planted in

the trays. Three replicates were made for each treatment.

Results. The results are given in Table IT.

 Table 17. Effect of sterilization of mummy infusions

and filter pads on the ability to induce abnormalities

Treatments Replication Results

Mummy soil infusion 1 OP

2 np 13 ov
3 np 3 ov

Mummy compost infusion 1 np

2 np
3 np

Mummy soil infusion autoclaved 1 np

2 np
3 np-

Mummy soil infusion filtered 1 np

2 np
3 np

Mummy compost infusion autoclaved 1 np

2 np
3 np 5 ov

Mummy compost infusion filtered 1 np

2 np
3 np

Mummy soil infusion filters 1 np

2 np
3 np

Mummy compost infusion filters 1 np

2 np
3 np 25 ov

This experiment had been planned with the expectation that the

mummy soil water infusions and the mummy compost water infusions

would induce the production of open-veiled mushrooms as they had in

Experiment 5 (Table 13 )# Since this did not occur, no conclusions

can be drawn from the results of the sterilization treatments.

 47.

Experiment 10.

Methods, The purpose of this experiment was to determine

if the mummy disease infecting white mushrooms could be transmitted

to cream mushrooms.

The mummy soil materials and the, mummy compost materials used

in this experiment were obtained from the bed of Experiment 4 which

had been spawned with The Pennsylvania State University white strain

286. Two trays of each of the following cultures were infested with

soil and compost as in Experiment 1: Pennsylvania State University

white strains 286, 282, and 296; Pennsylvania State University cream

strains 301 and 308; Butler County Mushroom Farm's golden-white D-26.

Results. The results are given in Table 18.

 48*

Table 18. Effect of infecting different strains of

Agaricus campestris with soil and compost taken from

areas producing mummy in a white strain (286)

Treatment
Strain Control Infested

White
286 np 1 * mummy
2 0 mammy

282 np 1 „ ismsay
2 . naimy

296 np 1 . mummy
2 . mummy

Golden white
D-26 np 1 * np
2 . np

Cream
301 np 1 * np
2 „ np

308 np 1 . .np
2 . np

Although the number of replications of each treatment in

Experiment 10 does not warrant definite conclusions# there is an

indication that the mummy disease in a white strain of mushroom could

not be transmitted to a non-white strain but could be transmitted to

other white strains.

Experiment H *

f»iethods. The purpose of this experiment was to determine

if any of three fungi isolated in Observation 5 and associated with

mummy or open-veil materials could produce any abnormalities when

 49*

placed in an otherwise untreated tray* The three fungi* a Dictyo-

stelium sp,f a Fusarium sp*, and an unidentified phycomycete which

was either a Phytophthora or Pvthium species, were grown on potato-

dextrose-yeast agar slants for three weeks at room temperature* Two

slants of each isolate were then placed into recently cased trays so

that the fungus was in contact with the soil and the compost* This

treatment with each isolate was replicated three times*

Results, None of the trays infested with the three fungi,

a Dictyostelium sp*„ a Fusarium sp*, and an unidentified phycomycete,

produced any abnormalities during the cropping period*

Experiment 12*

Methods* The purpose of this experiment was to determine

if the nematodes obtained in Observation 15 could produce any ab­

normalities when placed into normally producing trays* Each Gf

three normally producing trays were infested with 10,000 nematodes

suspended in 100 ml* of water* The suspension was poured into the

trays so that the nematodes would contact both the soil and the

compost*

Results* None of the trays infested with the nematodes

obtained in Observation 15 produced any abnormalities during the

cropping period*

Experiment 13*

Elethods* The purpose of this experiment was to determine

if the infective agent could be rendered non-infective by subjecting

compost and soil from an affected area to heat treatment* Soil and

compost from an affected area in a mushroom house at Temple,

Pennsylvania, were brought to The University in fifteen-gallon

galvanized cans and stored at 50° F, for one week* Soil or compost

were placed into 100 mm* diameter covered glass moist chambers to a

depth of one-half to three-quarters of an inch and subjected to

temperatures of 40° C OB 60° C , , and 100° C, for 15e 3O 0 45, and 60

minutes* The 40° C» and the 60° C, treatments were obtained by

immersing the moist chambers in a water bath of the respective

temperatures, plus or minus one degree Centigrade, The pre-heating

period for either temperature was twenty minutes for the soil and

thirty minutes for the compost as determined in trial runs. Treat­

ments at 100° C, ware obtained by placing the moist chambers in an

autoclave with flowing steam. Timing began when the temperature on

the exhaust line of the autoclave reached 100° 0 o Soil or compost

from three moist chambers were combined to treat one tray in the

growing house. Three replicate trays were provided for each

treatment plus control trays treated with soil and/or compost from

an affected area.

Results, None of the 84 trays of Experiment 13 produced

any symptoms of the mummy disease or any other abnormalities. Since

the control (infested) trays did not show mummy symptoms, no con­

clusions can be drawn regarding the trays infested with soil or

compost which had been subjected to the thermal treatments. This

failure, for the first time, to transmit the mammy disease from in­

fested soil and compost, could not be explained by any facts known

about the disease and its behavior.

 VII. DISCUSSION

Correlation between commercial growing practices and the occurrence

of the mummy disease.

No correlations could be found between any commercial growing

practices examined and the occurrence of this disease. This may have

been because none of the practices recorded had any relation to the

disease or because quantitative data regarding these various prac­

tices could not be obtained. Tucker and Eoutien (8 ) indicated that

the disease usually appeared at random in the growing area and did

not necessarily occur in the same area in subsequent crops, and

never appeared before the third break. The information obtained in

this study agrees with Tucker and Routien’s findings on the first

two points, but the disease appeared as frequently on the first and

second breaks as it did on the third. The appearance of the disease

on beds cased with soil treated with steam and formaldehyde does not

support Tucker and Routien*s hypothesis that the disease might have

been introduced into the growing area by the use of untreated casing

soil, however, there is a possibility that the steam and formaldehyde

treatment may not have been sufficiently thorough in all cases to

eliminate the causal factor.

Hicroflora and taicrofauna

At the beginning of this study, the possibilities of the mummy

disease being caused by an outside agent, such as a virus, a

bacterium, a fungus, a protozoan, a higher animal, or a combination

of these, were considered. Bacteria were dropped from the suspect

list since the disease coaid not be transmitted by infusions from

soil or compost or by the bacterial filters through which these in-

S

fusions had passed. Bawden and Gregory (2) and Storey (4) attempted

to test Tucker and Routien's virus hypothesis and obtained nega­

tive results. This present study concentrated on isolating a fungus

which might not sporulate in the compost or soil* or a transient

animal not noticed by Tucker and Routien. Five organisms, a yellow

Trichoderma viride. a Fusarium sp.# an Acremonium sp., a Dictyo-

stelium sp., and an unidentified phycomycete were isolated from mummy

materials but not from corresponding normal materials. None of these

organisms could induce the mummy disease ivhen normal mushroom trays

were infested with them although control infestations produced the

mummy disease. No attempts were made to re-isolate the organisms

and it is not known if they actually became established. Tucker and

/

Routien (8 ) isolated a Trichoderma sp.f a Fusarium sp,* and a Pvthium

sp, from the basal region of the stipe of mummy mushrooms and could

not induce the mummy disease by infesting beds with these organisms.

The compost and soil of all normally producing, open-veiled

mushroom producing and mummy producing trays were examined. No ob­

served organisms could be associated with the open-veil producing

trays or the mummy producing trays. Tucker and Routien (8 ) found the

same situation and observations of mummy infestations in commercial

growing plants indicated that no observable organisms were associated

with the mummy disease.

The nematode population of soil and compost in commercial

growing plants appeared to be similar for normal compost and soil and
compost snd soil in an affected area* The presence of nematodes was

not a prerequisite for the appearance of disease symptoms0 A popu­

lation count of nematodes (Observation 15) during Experiments 5 and 6

indicated that they may have some part in inducing the disense9 but in

in an infestation experiment (Experiment 12) with these same nematodes

no trays produced mummy mushrooms,. The nematodes were probably

introduced into the mummy producing trays incidentally during the

infestation procedure.

No protozoa could be found in mummy soil 'which were not present

in normal soil, and no protozoa were found in either normal or

affected compost in Observation 16, During the course of this study*

a great many samples of soil and compost were examined at various

magnifications and no animal organisms were ever found in mummy

materials which were not also present in normal materials#

The nature and habitat of the causal organism

Tucker and Routien (0) found that the disease could be trans­

mitted to normally producing beds or frays by transferring soil or

compost from within diseased areas or from areas adjacent to mummied

w e n s into the normal beds or trays. Experiment 1 confirmed their

res?* It s., The ability to transmit the disease in this manner indi­

cated that a nutritional or environmental factor was not the primary

-.gent involved, The spread of the disease through two infested beds

■'Experiment d) substantiates this conclusion.

Soil and compost from affected areas treated with propylene

oxide could not transmit the disease. Tucker and Routien (0) found

that heat sterilization rendered soil and compost from affected areas
 54*

non-infective* and this experiment indicated that it was the steri­

lization that was pertinent and not the heat per se.

Water infusions made from soil and compost from affected areas

could not transmit the disease (Experiment 2), but a later experiment

(Experiment 6 ) indicated that these infusions induced the production in

moderate numbers of open-veiled or hard-gilled mushrooms* Since

open-veiled mushrooms had not appeared in any previous experiments

of any type in the experimental house* it was postulated that some

component of a complex causing the mummy disease was separated from

the soil or compost by the infusion process. Experiments 7 and 8

were designed to test this hypothesis* that is, to determine if

mummy could be induced by combinations of infusions and leached

materials* and that the open-veil condition was not self-per­

petuating, The occurrence of the open-veiled mushrooms during these

experiments could not be associated with the mummy disease and was

probably an incidental phenomenon, Pn experiment designed to test

sterilized infusions also supports this conclusion.

The mummy disease affecting the white variety of the cultivated

mushroom apparently can not be transmitted to non-white varieties.

This phenomenon might explain the failure in Experiment 3 to trans­

mit the disease with soil and compost from an affected area. Tucker

and Routien (8 ) had observed that both the white and cream varieties

of the cultivated mushroom are susceptible to the mummy disease* but

they did not attempt to infest one variety with materials from the

other variety. This limited transmissibility indicates that it is

important in future studies to use the same strain when doing

 55,

infestation experiments. The possibility of using this varietal

response may be a tool for further research. It may be possible to

determine if the infectivity is dependent on the ability of the

mycelium of one variety or strain to anastomose with the hyphae of

another variety or strain.

Since the mummy disease could not be transmitted without trans­

ferring living mycelium of the mushroom, the causal agent was probably

intimately associated with and dependent upon the Aqaricus campestris

mycelium. This fact led Tucker and Routien (8) to suggest the virus

hypothesis. However, it would appear that any non-sporulating

organism which would be an obligate parasite would also adhere to

these conditions. In this study, and apparently in the work of

Tucker and Routien (8), the mycelium of Aqarieus campestris was never

isolated from infective materials and consequently such an organism

would not have been observed. It would appear that further investi­

gations on this disease should concentrate on the isolation and test-

ting of mycelium from materials proven infective, but even work of

this type might prove misleading if the isolated mycelium were non-

infective and the hyphae which could not be isolated were infective.
 VIII, SUMMARY

A study of the mummy disease of the cultivated mushroom,

Aqaricus campestris L„ ex Fries, was initiated for the purpose

of identifying the causal agent or obtaining information about the

disease which could assist subsequent investigations,,

Commercial mushroom plants in which the disease appeared were

visited in an attempt to find some common practice which may have

been responsible for the introduction of the disease, Normal and

infested samples of compost and soil were examined to determine if

there were any differences in their microflora or microfauna,

Various transmission experiments were conducted in order to establish

the nature or habitat of the causal agent.

No correlation was obtained between any commercial growing

practices and the occurrence of the mummy disease,*

Five fungi, Trichoderma viride a Fusarium s?; ««

sp,e a Dictyostelium sp,, and an unidentified phycomycete nere

not found in normal compost or soil were isolated from mummy infested

compost or soil,, None of these organisms could induce the production

of the mummy disease when planted in normal mushroom trays.

The xieraatode population of soil and compost of infested areas

appeared to be the same as normal areas. Trays infested with nema­

todes obtained from soil anu compost of affected areas produced no

abnormalities.

An Initial experiment indicated that water infusions obtained

from soil and compost of affected areas could induce the production

of open-veiled mushrooms,, This could not be substantiated in later

 57 „

experiments.

Treatments of soil or compost from affected areas with propylene

oxide rendered them non-infective.

The disease could not be transmitted to golden white or cream

mushrooms by the infestation of soil and compost which did transmit

the disease to three white strains.

An experiment to determine the thermal inactivation time was

not successful since neither controls nor treatments became diseased,,

 LITERATURE CITED

Atkins, F, C., 1956, Diagnosis of mummy, M.G.A. Bull. 74:44-46*

Bawden, F. C., and R, H. Gregory. Rothamsted Exp. Sta. .Report for

1951. pp. 84-85.

Fleury, C.t 1948. Rev. Horticol. Suisse 21:279-282.

Storey, I. F., 1954. Mummy disease of mushrooms. Plant

Pathology 3:49.

Thomas, C. A., and G„ 1-1. Mitchell, 1950. Eelworms (Nematodes)

as pests of mushrooms. M.G.A. Review 16 pp.

Tucker, C» M., 1937. A destructive disease of mushrooms. Proc.

Mo. Acad. Sci. 3:71-72 (Abstract).

Tucker, C. M., and K. IV. Simons, 1940. A disease of cultivated

mushrooms. Mo. Agr. Exp. Sta. Bull. 413:32^34.

Tucker, C. M., ar*d jc B. Routien, 1942. The mummy disease of

the cultivated mushroom. Moc Agr. Exp. Sta. Res. Bull. 358.
 APPENDIX

Cooperating Mushroom Growers

Robert Adams, Temple, Pennsylvania

Breezyacres Mushroom Farm, Temple, Pennsylvania

Brandywine Growing Corporation, Nest Grove, Pennsylvania

Butler County Mushroom Farm, West Winfield, Pennsylvania

John Caliguiri, Mt* Pocono, Pennsylvania

Joseph Caliguiri, Mt, Pocono Pennsylvania

Ettore Catnilli and Sons, Temple, Pennsylvania

Frank Evanno, Temple, Pennsylvania

Raymond Gaspari, Temple, Pennsylvania

William Ghione, Kennett Square, Pennsylvania

Armando Morganti, Temple, Pennsylvania

Walter Needham, Avondale, Pennsylvania

Penn Mushroom Company, Temple, Pennsylvania

William Saganich, Mt„ Pocono, Pennsylvania

 Vita

Edward L. Merek was born in Philadelphia, Pennsylvania on

January 16, 1930„ He was graduated from Central High School of

Philadelphia in 1947, and received a Bachelor of Science degree

in Botany in 1951 and a Master of Science degree in Botany in 1953

from The Pennsylvania State University, University Park, Pennsylvania*

He served two years in the Army Chemical Corps, United States Army*

He is a member of the Mycolorjical Society of America,,