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The occurrence and effects of Cephlothecium roseum on the avocado

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THE OCCURRENCE AND EFFECTS OF CEPHALOTHECIUM ROSEUM ON THE AVOCADO

A Thesis Presented to The Faculty of the Department of Botany The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by John W* Yale, Jr* February, 1950

UMI Number: EP70611

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

UMT D issertation Publishing

UMI EP70611 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code

Pro ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 481 06- 1346

This thesis, w ritte n by

under the guidance o f

AAa... F a c u lty C o m m itte e,

and app ro ved by a l l its members, has been presented to and accepted by the C o u n c il on G raduate S tudy and Research in p a r t ia l f u l f i l l ­ ment of the requirements f o r the degree o f

12&.CL

Faculty Committee

Chairman

ACKNOWLEDGEMENT The author wishes to express his thanks and appreciation for the assistance and guidance obtained from all three members of the committee* Grateful acknowledgement is made to Dr. George B. Johnstone for his patience and thoughtful understanding while supervising the progress of this investigation.

Appreciation

is due Dr. Louis C. Wheeler for devoting his time to taking color photographs and for making suggestions and helpful criticism in the prepara­ tion of this thesis.

Appreciation is accorded

Dr. Tilden W. Roberts for his advise and help in taking photomicrographs and for the encouragement he afforded throughout this study. Thanks and gratitude are expressed to Dr. Thomas C. Fuller for his assistance and guid­ ance in preparing sections of infected avocado stems.

ii

TABLE OF CONTENTS SUBJECT

PAGE

INTRODUCTION......................

1

HISTORICAL REVIEW OF CEPHALOTHECIUM R O S E U M .........

2

M A T E R I A L S .........................................

6

METHODS

..............................................

7

Isolation of C. r o s e u m .........................

7

Observation of identifying characteristics.. .......

8

Establishment of parasitism on avocado fruits,. . .

8

Establishment of parasitism on avocado stems • • • •

10

Environmental relations of C. roseum ♦

12

..........

Method for testing the susceptibility of different varieties

• • •

18

..............

General laboratory techniques. • • • • • • • • • • • 19

RESULTS OF THE INVESTIGATION....................... C. roseum on avocado fruits.

18

..................

19

C. roseum on avocado s t e m s .....................

27

Et iology.................... Environmental relations of C . r o s e u m ........... Control of C. roseum on avocado fruits and stems

37 ¥+ . •

SUMMARY.............................................

6l

BIBLIOGRAPHY.......................................

66

APPENDIX

57

77

LIST OF TABLES TABLE I. II. III.

PAGE Hosts of Cephalothecium roseum

Substrata Favorable to C. roseum • • • • • • • • .

5

............ I1**

Frequency of Occurrence and Aggressiveness of C. roseum on Market F r u i t s ..........• • • • • •

V.

3

Relative Humidity Regulation by Calcium Chloride Solutions, (Smith, G., 19*+6)

IV.

...........

22

Cephalothecium roseum, Rhizopus nigricans. Cladosporium sp., and Acrostalagmus sp., in the Order of Their Decreasing Aggressiveness on Fifty-Six Decaying Fuerte Avocados ............

VI.

Manner in which Cephalothecium roseum Infects Healthy Avocados

VII.

..........

Substratum— avocado................. k6

Substratum— maltose peptone agar . •

b-?

Effects of Relative Humidity upon the Growth of C. roseum.

Substratum— Sabouraud1s dextrose

peptone a g a r ................ XI.

35

Effects of Relative Humidity upon the Growth of C . roseum.

X.

25

Effects of Relative Humidity upon the Growth of C. roseum.

IX.

........................

Artificial Inoculation of Avocado Stems in the Orchard.

VIII.

23

^8

Effect of Constant Light Intensity upon the Growth of C. roseum

52

iv

v LIST OF TABLES (Continued) TABLE XII.

PAGE Susceptibility of Five Different Varieties of Avocados to Infection by C. r o s e u m .............. 55

XIII.

Four Spoilage Fungi in the Order of Their Decreas­ ing Aggressiveness on Fuerte Avocados. • . • • •

XIV.

Manner in which £. roseum Infects Healthy Nabal Avocados • • • • • • •

XV. XVI.

77

..............

. . • • •

73

Cultural Data from Le Clerg, 1930...........

79

Fungi which Infect Avocado Fruits. • • » • • • • •

8l

LIST OF FIGURES FIGURE 1*

PAGE

Apparatus Used to Regulate the Relative Humidity In a Closed C h a m b e r

2.

. . . . . . .

15

Apparatus Used to Expose Cultures of C. roseum to a Constant Light Intensity and Temperature . .

17

3.

Occurrence of C. roseum on a Fuerte Avocado . . . .

20

h.

Wounded Fuerte Avocados Infected with C. roseum . •

28

5.

Cross-section of Fuerte Avocado Infected with C. r o s e u m .........................................28

6.

Nabal Avocado Infected with C. roseum .

.......... 30

7.

C. roseum on Young Fuerte Avocado Stems . . • • • »

8*

0. roseum on a Dead Twig Taken from the Litter beneath a Fuerte Avocado T r e e ..........

9.

................• • • • • • • • . .

32

Longitudinal Section Showing Hyphae of C. roseum in Vessels of an Avocado Stem • • • • • • • • • .

11.

31

Transverse Section of Avocado Stem Infected with C. roseum

10.

31

33

Transverse Section Showing Hyphae of C. roseum in Vessels of an Avocado Stem • • • • • • • • • •

12*

£* roseum:

Living, Slide Culture • • • • • • • • •

13.

Beginning of Conidial Cluster at Tip of Conidiop h o r e .......... • • • • ...............

33 **1

**1

l1*.

Formation of Conidia of C. r o s e u m .................. **2

!?•

£• roseum: Germination of Conidia vi

..........

^3

INTRODUCTION Present status of our knowledge.

Cephalothecium

roseum Corda occurs as spoilage organism on avocado fruits and as a faculative saprophyte on avocado stems. The literature concerning the occurrence and effects of Cephalothecium roseum on the avocado is limited to an acknowledgement by William T. Horne, 193*+* that a species of Cephalothecium occurs on old decayed fruits, and that it is often mistaken for Colletotrichum gloeosporioides which causes anthracnose rot of avocados.

Both organisms produce

a powdery, pink, spore mass on the surface of decaying fruit (Horne, 193^)• Statement of the problem.

The purpose of this inves­

tigation is to determine first, the frequency of occurrence and the relative aggressiveness of Cephalothecium roseum on avocados at the market, the manner in which the fruit is infected, and to what extent the fruit is damaged. The second objective of the investigation is to demon­ strate the occurrence of C. roseum as a facultative saprophyte on avocado stems, and to determine the distribution of the fungus on avocado stems in the field. The final objective is to suggest a means of control by observing and studying the effects of the environmental influences upon the growth and vigor of C. roseum. both in the field and in the laboratory. 1

HISTORICAL REVIEW OF C. ROSEUM A review of the history of C. roseum will serve as an index to the type of organism being investigated. C. roseum was discovered in Austria during the first half of the nineteenth century and is now known all over the world (Corda, IB385 Link l82*f; and Hesler & Whetzel, 1917). It is one of the most common pink molds encountered, and occurs widely upon a great variety of hosts and substrata. Its conidia may be found floating in the air almost anywhere (Wolf, F. T., 19^3). The best known disease caused by C. roseum is pink rot of apples.

A major part of the literature concerning

C. roseum is concerned with this disease, which assumed great importance when the practice of piling and sweating apples was in vogue.

The years 1882, 189^, and 1902, stand

out in the history of the apple industry in western New York because of the epiphytotics of pink rot.

Authorities esti­

mated that thousands of barrels of apples were destroyed in one year in New York State by pink rot.

Experiences have

been similar in Ohio, Michigan, and Nebraska (Hesler & Whetzel, 1917). Some of the hosts of C. roseum reported in the litera­ ture are presented in Table I, along with the author and year of the publication.

Various substrata on which C. roseum has

2

3 TABLE I HOSTS OF CEPHALOTHECIUM ROSEUM

Host

Year

Cause

Author

1 .

Melons

1911

Laubert

!• roseum

2.

Pears

1917

£• roseum

3.

Quince

1917

Hesler & Whetzel If

b.

Grapes

1917

It

5*

Plum

1917

tt

£• roseum

6,

Egg Plant

1927

M, Lawson

C. roseum

7.

Lima Bean

1927

tt

£• roseum

8.

Summer Squash

1927

tt

9.

Cucumber

1928

tt

1930

0, Verona

2* roseum

Vanderwalle

2* roseum

c .

roseum

£• roseum c .

roseum

Apples

11.

Smut Fungi

1932

CM H

Ten var. of apples, S, Am, Avocado

1931*- Haussmann 193^

Horne

£•

1

• —I

Muskmelons

1936

Wiant

£• roseum

15.

Castor Bean

1937

Golovin

2* roseum

16.

19*+3

Heald

£• roseum

17.

C onidia-bearing stromata Pythium Oospores

19^3

Lrechsler

2* roseum

18 ♦

Sweet Potato

19^9

Nesbitt

£• roseum



10,

13.

&

Pears

£• roseum

2* roseum S

£.

* T. roseum (Trichothecium roseum Link), is regarded as being synonymous with Cephalothecium roseum Corda, See Etiology, p. 37.

been found growing are presented in Table II*

References to

these publications may be found in the literature cited* C. roseum is most commonly encountered in fruit and vegetable produce markets where the organism is responsible for the spoilage of many fruits and vegetables (Ramsey, G. B 1938; Link, G. K. K., 1919; and Dawson, M* L., 1931). The wide and varied number of hosts and substrata which favor the growth of C* roseum indicate the importance of this facultative saprophyte (Simonet, 1926). Further correlation of the literature will be found in the methods and results of the investigation.

5

TABLE II SUBSTRATA FAVORABLE TO CEPHALOTHECIUM ROSEUM

Substrata

Year

Author

Cause

1.

Various leaves and twigs

1925

Stevens, F. L.

C. roseum

2.

Wool fabric previously decontaminated by dicont. steaming

1928

Burgess

C. roseum

Honey-dew droppings from aphids on cotton bolls 1929

Nikolsky

!• roseum

Wool treated with alkali 1929

Burgess

c. roseum

5.

Butter

1930

Grimes

2* roseum

6.

In cans of petrol con­ taining pieces of leaves

1930

Verona

2* roseum

7.

Soil— irrigated and non-irrigates

1930

LeClerg

c. roseum

8.

In human stomach

1939

Ferrando

!• roseum

9.

Avocado branches

19^8

The author

c. roseum

H O •

3.

Dairy & Rabbit manure

19^8

The author

c. roseum

11.

Dead apple branches

19*+9 The author

c. roseum

12.

Trunk of Dracena draco

19*+9

The author

c. roseum

MATERIALS The avocados used to determine the frequency of occur­ rence and the relative aggressiveness of Cephalothecium roseum on market fruits were obtained from the Alpha Beta Food Market, Fullerton, California. The fruits used to make artificial inoculations were obtained from two main sources.

The Hass, Nabal, Aneheira,

and Harmon varieties were purchased from the Table Praise Avocado Co., Yorba Linda, California.

The Fuerte fruits were

donated by John W. Yale, avocado rancher, Yorba Linda. Avocado stems infected with C. roseum were collected from ranches in the Yorba Linda, Placentia, and La Habra areas of southern California. Fuerte avocado trees, owned by John W. Yale, Yorba Linda, were used in the experiments which required the inocu­ lation of fruits and stems still attached to the tree. The laboratory and photographic equipment were supplied by The University of Southern California.

6

METHODS THE ISOLATION OF CEPHALOTHECIUM ROSEUM C . roseum was first observed on avocados obtained from the Alpha Beta Food Market, Fullerton, California.

The fungus

was isolated by removing small portions of the mycelium from the infected fruit and placing them on a favorable, decontami­ nated agar medium. Single spore isolations were prepared in the following manner.

A small piece of mycelium was removed from a culture

of C. roseum and mixed with 25 milliliters of boiled, distilled water.

A few drops of this mixture were removed with a decon­

taminated pipette and diluted with another 25 milliliters of boiled, distilled water.

A few drops of this dilution were

spread out over the surface of a thin, decontaminated layer of nutrient agar contained within a Petri dish.

After two days

incubation at room temperature, the spores were observed under the microscope.

The well isolated spores which were beginning

to germinate were removed from the agar plate by lifting the sporling and a small bit of the agar beneath it with the flattened end of a thin piece of wire.

These single sporlings

were placed in separate test tubes which contained decontami­ nated, nutrient agar slants.

These isolates of C. roseum

were stored in a dark closet to be used in the artificial inoculation experiments conducted later.

7

8

OBSERVATION OF IDENTIFYING: CHAftACISRISXICS The morphological characteristics used to identify C.

roseum were observed by growing the fungus on slide cul­

tures.

The growth and development of C. roseum were studied

under the microscope without disturbing the fungus or con­ taminating the culture with other organisms. The slide cultures were prepared in the following manner.

Thin cardboard was cut into strips one sixteenth of

an inch wide, and as long as the side of a square cover glass. For each slide culture three cardboard strips were used.

The

strips were dipped in thin balsam, and arranged on a glass slide in the form of a square with one side missing.

A few

drops of nutrient agar were placed between the cardboard strips on the glass slide and inoculated with a bit of mycelium and conidia taken from an isolate of C. roseum.

The fungus cul­

ture was enclosed in a low chamber by placing a square cover glass on top of the cardboard strips. The slide cultures were incubated in Petri dishes which had moist filter paper in the bottom. ESTABLISHMENT OF PARASITISM ON AVOCADO FRUITS Under certain conditions healthy avocado fruits may be infected by C. roseum.

To determine these conditions, the

following inoculation tests were made.

9 Artificial inoculation of harvested fruits.

Twelve

freshly harvested Fuerte avocados, firm, and of approximately equal size and degree of maturity, were decontaminated in 95 per cent ethanol, and then rinsed in boiled, distilled water.

The fruits were free from any wounds, until the

pedicels were removed, exposing the pedicel scar pit.

A

conidial water suspension, prepared from an isolate of C. roseum. was used to inoculate the fruit. Four of the fruits were inoculated with a drop of the conidial suspension placed on the unwounded surface.

Two of

these fruits were incubated in moist chambers, and the other two were left exposed to the relative humidity of the labora­ tory. Four other fruits were inoculated with a drop of the conidial suspension in an X-cut wound through the cuticle. Two of these fruits were placed in a moist chamber, while the other two remained exposed to the atmosphere of the laboratory. The last four fruits were inoculated with a drop of the conidial suspension placed in the pedicel scar.

These

fruits were divided into moist and open chambers as before. The twelve fruits were incubated for nine days at room temperature. The results of these inoculations are found in Table VI, and discussed on page 2*f.

10 When this experiment was repeated using Nabal avocados the same results were obtained*

(See Appendix, page 78).

Artificial inoculation of unharvested fruits*

To

determine if avocados can become infected with C. roseum while still hanging on the tree, the following inoculation tests were made. Six large Fuerte avocados, attached to the same tree and situated in a shaded, protected, part of the tree, were inoculated with a water suspension of conidia made from an isolate of C. roseum.

The fungus was inoculated into a

wound made on the side of the fruit.

Three of the fruits

were covered with waxed paper bags to insure a high relative humidity about the fruit.

The other three fruits were left

exposed to the atmosphere. The results obtained from this experiment are found on page 2*f. ESTABLISHMENT OF PARASITISM JQfi AVOCADO STEMS The following methods were employed to determine the nature of the association of C. roseum on living avocado stems. Artificial inoculation of living stems.

Two healthy,

green stems, of approximately equal size, were selected on five different Fuerte trees.

A smooth surface on each of

11 the ten stems was decontaminated with 95 per cent ethanol, rinsed with boiled distilled water, and dried with sterile cotton.

Three small circular wounds were made about three

inches apart on each decontaminated surface. wound on each stem was left decontaminated.

The first The second

wound was inoculated with a conidial water suspension pre­ pared from an isolate of C. roseum.

The third wound on each

stem was inoculated with a solution of unknown toxicity pre­ pared from a nutrient agar medium upon 'which C. roseum had been grown for four weeks.

The lower half of the medium,

which was free from any hyphae or conidia, was separated from the upper half.

This lower half of the medium was placed

in enough boiled distilled water to cover it, and left for two days.

It was assumed that any toxic substance produced

by the fungus would be diffused throughout the agar medium and would diffuse out into the water surrounding it.

This

solution of unknown toxicity was used to inoculate the third wound on each stem. The stem wounds were carefully covered with waxed paper bags to insure a favorable relative humidity about the wounds and to prevent contamination from air-borne disseminules. The results obtained from this experiment are presented in Table VII, and discussed on page 29. Mounting sections of infected stems.

Living, green

12 steins, about one-half to three-quarters of an inch in diameter, and infected with C. roseum, were collected from an avocado tree in Yorba Linda.

Small blocks of the infected tissue

were imbedded in cellodion according to the schedules described by Johansen, 1 9 ^ , and Blaydes (unpublished).

They were

mounted upon hardwood blocks and sectioned with the sliding microtome.

Transverse and longitudinal sections, twenty

microns thick, were cut.

The cellodion was removed from

the sections with 50-50 ether and absolute alcohol.

They

were stained with picroanalin blue and safranin, dehydrated from 95 to 100 per cent alcohol, cleared in clove oil, rinsed in xylol, and mounted in balsam.

The lignified cell walls

stained red, the cellulose walls of parenchymatous cells stained blue.

The hyphae of the fungus were brown and stood

out distinctly in the stem tissue. The results obtained from these infected stem sections are presented in Figures 9* 10, and 11, and discussed on page 29. ENVIRONMENTAL RELATIONS OF C. ROSEUM The following methods were employed to test the effects of environmental influences upon the growth and virulence of C. roseum. Regulation of relative humidity.

By regulating the

relative humidity of closed chambers in which C. roseum was

13 growing, the effects upon the degree of sporulation and the rate of vegetative growth could be observed.

For this experi­

ment, Ball, Eclipse, wide-mouth, pint jars with rubber gaskets, glass lids, and wire fasteners were used for chambers in which the relative humidity could be regulated. stoppers

Inverted glass

(Figure 1) were used to hold the substrata on which

C • roseum was to be grown. The relative humidity was regulated by adding calcium chloride solutions of varying concentration to the chambers. The relative humidities were determined by the conditions given in Table III. The apparatus used for the regulation of the relative humidity in closed chambers is illustrated in Figure 1. The temperature was kept at 20°C. by placing the incubation chambers in a regulated hot water bath.

The hot

water bath was in turn placed in a constant temperature room, o maintained at lp C. The substrata were inoculated with a conidial suspen­ sion from an isolate of C. roseum and allowed to develop in the regulated humidity chambers for seven days. The results of the experiments, in which the effects of the relative humidity upon the growth and virilence of C. roseum were tested, are presented in Tables VIII, IX, and X, and discussed on page M+.

11+

TABLE III RELATIVE HUMIDITY REGULATION BY CALCIUM CHLORIDE SOLUTIONS (SMITH, G. , 19^6)

Gm. of CaClg.ffigO in 100 gm. wateF

Density in gm./ml. at 15°C.

Relative Humidity in % at 20°C.

15

1.115

89.7

20

1.150

87.5

30

1.213

77.b

>*0

1.272

66.3

50

1.325

55.3

60

1.371*-

^3.9

70

1.396

3*f.l

15

.CUUTUHE KiDltifc .

FIGURE 1 APPARATUS USED FOR THE REGULATION OF RELATIVE HUMIDITY IN A CLOSED CHAMBER

The regulation of light intensity at a constant tempera­ ture,

To determine the effects of light intensity upon the

rate of growth of C. roseum, the following experiment was conducted.

Eight Petri dishes, each containing a corn dex­

trose agar medium, were inoculated with one drop of a water suspension of conidia from C. roseum.

Four of the dishes were

placed in a dark chamber, so that the fungus could develop in the absence of light.

The other four Petri dishes were exposed

to a constant light intensity of 230 ft. candles. tus for this experiment is illustrated in Figure 2.

The appara­ A five-

hundred watt incandescent light bulb was suspended in a battery jar.

Flowing water through the battery jar absorbed

the heat given off by the light bulb.

The four fungous cul­

tures were placed a few inches beneath the bottom of the battery jar.

The continuous flow of water between the light

and the fungous cultures kept the temperature regulated to an average of 23.9°C.

The temperature in the dark chamber was , o maintained at an average of 24.0 C. The temperature was measured by placing maximum-minimum thermometers in the posi­ tions to be occupied by the fungous cultures.

After five days

of incubation, the diameter of each culture was measured. The results of this experiment are presented in Table XI, and discussed on page 51* The regulation of temperature.

The minimum tempera­

ture at which conidial germination begins was determined in

17

a-—

FIGURE 2 APPARATUS FOR THE REGULATION OF LIGHT INTENSITY AT A CONSTANT TEMPERATURE

18 the following manner.

Decontaminated agar plates were inocu­

lated with conidia taken from an isolate of C. roseum.

These

plates were placed in a temperature regulated refrigerator. Tests were made from 1 to 12 degrees Centigrade, with incuba­ tion periods averaging ten days for each temperature tested. The results of this experiment are presented on page 51. METHOD FOR TESTING T^g SUSCEPTIBILITY OF DIFFERENT VARIETIES The Nabal, Anaheim, Hass, Fuerte, and Harmon varieties were tested for any differences in their susceptibility to infection by C. roseum.

Three fruits of each variety, of

approximately equal ripeness and degree of maturity, were decontaminated in 95 per cent ethanol for twenty-five seconds and then rinsed in boiled distilled water.

They were inocu­

lated with a water suspension of conidia made up from an isolate of C. roseum. and then incubated in moist chambers at room temperature. The results of this experiment are presented in Table XII, and discussed on page 5^* GENERAL LABORATORY TECHNIQUES The phytopathological techniques employed in this inves­ tigation were adopted from Rawlins, 1933, George Smith, 1 9^, Ernst Bessey, 1935, and A. T. Henrici, 19^7*

19 RESULTS OF THE INVESTIGATION The results of the investigation are presented in the following manners

(1) C. roseum on avocado fruits,

(2) C. roseum on avocado stems, (3) the classification and morphology of the organism, (b) the environmental relations of C. roseum, and (5) a suggested means of control. C. ROSEUM ON AVOCADO FRUITS The fruit of the avocado is often subjected to infec­ tion by numerous fungi, (see Appendix, page 8l).

Many of

these fungi are secondary invaders which grow only as saprophytes on the decaying fruit 5 others are active para­ sites which will readily infect the healthy fruit.

Among

these miscellaneous fruit decays, C. roseum was observed to be one of the most frequently occurring fungi on harvested avocados, Figure 3 . Frequency of occurrence and relative aggressiveness on market fruit.

To determine the frequency occurrence of

C. roseum on market fruit and its relative aggressiveness in relation to other superficial fungi present, fifty-six overly ripe Fuerte avocados, obtained from the Fullerton, Alpha Beta Food Market, were placed in moist chambers and incubated at room temperature for eighteen days. tained two fruits.

Each moist chamber con­

Half of the fruit was collected and

FIGURE 3

THE OCCURRENCE OF C. ROSEUM ON A FUERTE AVOCADO (LIGHT COLORED AREA). BROWN MOLD AT THE STEM END OF THE FRUIT IS ACROSIALAGMUS ALBUS. THE COMMON PIN IS INSERTED FOR SCALE. XO.6.

21 incubated on February 1, 19*+9 > and the other half on February

16, 19^9• Many different fungi developed on the surface of the fruits, and when the presence of C . roseum was detected, its relative aggressiveness was compared to that of the other fungi present*

The results of this experiment are presented

in Tables IV and V. C. roseum developed upon 75 per cent of the fruit tested, and it was the most aggressive organism on 36 per cent of the fruit (See Table V). The four most prevalent fungi, in the order of their aggressiveness were, C. roseum* Rhizopus nigricans* Cladosporium sp., and Acrostalagmus sp. (See Table V). During the experiment, the following observation was made.

In four of the moist chambers, Rhizopus nigricans and

Cephalothecium roseum developed simultaneously upon the sur­ face of the fruit.

When the hyphae of both organisms met,

the hyphae of Rhizopus nigricans grew up over the growth of £♦ roseum and completely hid it from view. moist chambers the exact opposite occurred.

In three different The hyphae of

C. roseum completely enveloped the growth of Rhizopus nigricans.

Even the sporangiophores and the sporangia of

Rhizopus nigricans were covered with a compact growth of the pink mold.

This gave the appearance of a pink Rhizopus.

A

few black sporangia of Rhizopus nigricans pushed up through

22 TABLE IV FREQUENCY OF OCCURRENCE AND AGGRESSIVENESS OF C. ROSEUM ON MARKET FRUITS. TWO FRUITS IN EACH MOIST CHAMBER. 1-1^ COLLECTED FEBRUARY 1, 19^9; 15-28 COLLECTED FEBRUARY 16, 19^9.

Moist Chamber Number 1 2 3 b 5 6 7 8 9 10 11 12 13 lb 15

16

C. roseum Present Absent

19 20 21 22 2? 2b

25 26 27 28

D

X X X X X X

C C B C C X

X X

* A*

D

X X

D D

X X X X X

A D B X

17

18

Aggressiveness

D D

X X X X X X

C B C D X

D

X X X X

* A - Unaggressive; B - Fairly Aggressive; C - Very Aggressive; D - Most Aggressive

C D

23

TABLE V CSPHALOTHECIUM ROSEUM. RHIZOPUS NIGRICANST CLADOSPORIUM SP., AND ACRQSTALAGMUS SP., IN THE ORDER OF THEIR DECREASING AGGRESSIVENESS ON FIFTY-SIX DECAYING FUERTE AVOCADOS

Order of Decreasing Aggressiveness ** *** E A B C D C. roseum

35.7

R. nigricans

39.3

Cladosoorium

3-57

Acrostalagmus

3.57

25.0

3.57

7.15 10.7 1^.3 7.15

7.15

28.6

0

>+2.8

7.15

10.7

6*+.3

3.57

36.1

82.1

See Appendix, p. 77* Percentage of fruit tested where the fungus was the most aggressive. Percentage of fruit tested where the fungus was unaggressive or not present at all.

2b the mat of C. roseum. showing that Rhizopus was still active. The Infection of Healthy Fruit Means of penetration.

Artificial inoculation tests

made on harvested fruits (Methods, page 7)? demonstrated the manner in which C. roseum infects healthy avocados.

The

results of these tests are presented in Table VI, and show that the infection of healthy avocados by C. roseum is possible only through the exposed pedicel scar or through an open wound.

The organism cannot infect the healthy fruit

through an unbroken cuticle.

These results substantiate the

claims of Hesler & Whetzel, 1917? Stevens, F. L., 1925? and Simonet, 1926, that C. roseum is a wound parasite. Although not shown in Table VI, C. roseum infected the stem end of the fruits through the pedicel scar whether it had been inoculated there or not. The wounded avocados were infected only when incubated in moist chambers.

It was observed that when the inoculated

wounds were on the upper side of the fruit in the open cham­ bers, no infection occurred; but when the fruits were turned over so that the wound was against the bottom of the con­ tainer, infection occurred.

This increased humidity about

the wound was enough to allow the fruit to become infected. Infection of unharvested fruit. Artificial inoculation tests were made on unharvested fruits to determine if avocados

25

TABLE VI MANNER IN WHICH C. ROSEUM INFECTS HEALTHY AVOCADOS

Conidial Suspension:

No. of Fruit

Incuba­ tion Chamber

Pos. No Infection Infection

2

moist

X

2

open

X

2

moist

2

open

2

moist

X

2

open

X

On unbroken cuticle X

In X-cut wound X

In pedicel scar

26 can become infected with C. roseum while still hanging on the tree (Methods, page 9)* Three days after inoculation, the three avocados which had been enclosed in waxed paper bags showed symptoms of infection.

By the fifth day, a white prostrate mycelium

could be observed developing on the inside of the wound on two of the fruits.

By the sixth day, signs of the organism

could be seen in the wound of the third fruit.

The flesh in

the wounded areas turned black. The three inoculated avocados that had been left exposed to the atmosphere showed no symptoms or signs of infection after three weeks. It is concluded that avocados are weakly susceptible to infection by £. roseum while still attached to the trees, providing, there is a wound in the fruit, and there is suffi­ cient moisture in the air to favor the growth of the fungus. C. roseum has not been found occurring naturally on any avocados still attached to the tree. Signs and Symptoms The dry rot produced on the fruit is characterized first by the appearance of dark sunken areas on the surface of the infected fruit.

Under moist conditions the organism

develops a compact mass of white mycelium on the infected surface.

After a few days sporulation occurs and the organism

27 becomes a pale pink mass of condiophores and conidia.

The

outer edge of the growth is white; while the older growth in the center is pink (Figure *+). When the pedicel scar is infected, the compact mass of mycelium and conidia can be seen within the scar pit.

The

stem end surface of the fruit turns black as the rot advances through the flesh.

The flesh of the fruit turns dark with

the presence of the organism, showing necrosis of the flesh, (Figure 5).

Any infection of the seed was not observed. C.

BOSEUM ON AVOCADO STEMS

Natural occurrence as a facultative saprophyte. A physiological die-back of small branches and twigs occurs regularly in healthy avocado trees.

Usually the leaves drop

from these branches and they die back gradually from the tip to the point of attachment.

Sometimes this die-back occurs

rather suddenly and the small branches die with the leaves still attached.

These dead twigs may persist for some time

before abscission occurs.

Oftentimes the die-back will begin

as a black, cankerous lesion anywhere on a green stem and progress both ways.

These cankerous lesions do not girdle

and kill the entire stem, but they travel up only one side, killing about half of it. C. roseum occurs as a saprophyte on the dead branches and twigs which persist in the trees and on those which are

FIGURE WOUNDED FUERTE AVOCADOS INFECTED WITH CEPHALOTHECIUM ROSEDM. XO.J.

FIGURE 5 CROSS-SECTION OF FUERTE AVOCADO INFECTED WITH CEPKAT.OTHF.CTUM ROSEUM. NOTE THE NECROSIS OF THE FLESH BELOW THE GROWTH OF THE FUNGUS. X 1.0.

29 found in the litter beneath the trees, (Figure 8). also occurs as a parasite on living stems.

The fungus

It is most com­

monly found on the small branches which are dying back from the tip, in cankerous lesions on green stems, in stem scars, or beneath the bark on larger limbs, (Figure 7)*

It has not

been found growing on avocado leaves, although F. L. Stevens, 1925, states that C. roseum has been reported growing on various stems and leaves from time to time. Establishment of Parasitism Transverse and longitudinal sections of infected tissue were prepared to verify the parasitism of C. roseum on living stems (Methods, p. 11).

The sections were made to

include the border line between the living and dead portions 0

of the infected stem.

Observation of the sections demon­

strated that the hyphae penetrate the living cells of the host (Figure 9)*

The hyphae appear to infect all of the

stem tissues, but are most concentrated in the vessels (Figures 10 and 11). After observing the parasitic nature of C. roseum on avocado stems, an experiment was conducted to establish whether or not C. roseum is responsible for the cankerous lesions or die-back of small avocado stems.

Young green

stems, attached to the tree, were inoculated in wounds with (1) a water suspension of conidia of C. roseum. and (2) a

30

FIGURE 6 NABAL AVOCADO INFECTED WITH C. ROSEUM. INFECTION BEGAN IN WOUND IN SIDE OF FRUIT AND IN STEM SCAR. X 0.5

31

i

A' ,, ■

■.

"J ■-'■.

v

B v

c ; *1

:

FIGURE 7 C. ROSEUM ON YOUNG FUERTE AVOCADO STEMS. A AND C, ON STEMS DYING BACK FROM THE TIPS. B, IN CANKEROUS LESION. X 1.0.

FIGURE 8 C. ROSEUM ON DEAD TWIG TAKEN FROM LITTER BENEATH FUERTE “ AVOCADO TREE. RUST-COLORED FUNGUS ON THE UNDERSIDE OF THE STEM IS ACROSTALAGMUS ALBUS. X 1.5.

FIGURE 9 TRANSVERSE SECTION OF AVOCADO STEM INFECTED WITH C. ROSEUM. PICTURE SHOVE BORDER LINE BETWEEN LIVING AND DEAD TISSUE. HYPHAE ARE PENETRATING LARGE PARENCHYMA CELLS OF PITH. X l*t0.0

FIGURE 10

FIGURE 11 LONGITUDINAL (UPPER) AND TRANSVERSE (LOWER) SECTION SHOWING HYPHAE OF C. ROSEUM IN VESSELS OF AVOCADO STEMl x I ki .O.

3b solution of unknown toxicity prepared from an artificial medium upon which the fungus had been growing (Methods, p. 8). A third set of wounds were left decontaminated for control. The results of this experiment are presented in Table VII. A few days after the inoculations were made, all three types of wounds became filled with a white sugary exudation. This phenomenon occurs regularly on wounded avocado stems. No sign or symptoms were observed for seventeen days.

On

the eighteenth day, four of the ten wounds that had been inoculated with C. roseum, began to show the following symptoms. The sugary exudation became watery and ran down the side of the stem.

Large amounts of this watery material were exuded from

the wounds for several days, and then the wounds became dry and there was no further exudation.

During this period, the

watery material was a pale pink as it exuded from the wound, and contained the hyphae and conidia of C. roseum. coloration was observed in the wound pit also. about two of the wounds became dark and sunken.

A pink

A small area Symptoms of

tissue breakdown were not observed in any of the other wounds. Eventually all but three of the wounds inoculated with C. roseum showed the symptoms described above. After the exudation had dried out there were no further evidences of infection.

The wounds that had not been inoculated

at all and those which had been inoculated with the solution of unknown toxicity did not show any symptoms of tissue breakdown

35

TABLE VII ARTIFICIAL INOCULATION OF AVOCADO STEMS IN THE ORCHARD INCUBATION PERIOD TWO MONTHS

Stem No.

Wound inoculated with C. roseum

Decontaminated Wound

Wound inoculated with solution of unknown toxicity

1

0

-

-

2

0

-

-

3

X

-

-

b

-

-

-

5

X

-

-

6

-

-

-

7

X

-

-

8

X

-

-

9

X

-

-

10

-

-

-

No symptoms. x Growth of C. roseum in wound, watery exudation. ~

and production of

o Growth of C. roseum in wound, production of watery exudation, and typical canker symptoms.

36 over a two-month period. The results of this experiment and the observations made of C. roseum occurring on avocado stems in the orchard (Figure 7) > indicate that the fungus is not the fundamental reason for the occurrence of stem die-back or the presence of cankerous lesions.

More likely die-back stems and can­

kerous lesions are the means of penetration for C. roseum, and the eventual death of host tissue in these areas may be further facilitated by the presence of this fungus. General Distribution C. roseum is distributed generally in Fuerte avocado groves in the Yorba Linda, Placentia, and La Habra areas of southern California.

Although a wide survey has not been

made, the results of this study indicate that it does not occur on the stems of all avocado varieties. been found only on Fuerte stems.

The fungus has

The stems of Puebla and

Nabal trees were conspicuously free from any growth of C. roseum in orchards where the stems of neighboring Fuerte trees were infected with the fungus. C. roseum can usually be found on five to ten trees per five acres of orchard if present at all.

The organism

cannot be found at all in some orchards, and in others it is present in at least one situation on almost every tree.

Some

trees may have only one branch infected, while other trees

37 may have many branches infected. It is concluded that C . roseum is scattered unevenly throughout Fuerte orchards in the areas mentioned, and that its frequency of occurrence is relatively small. ETIOLOGY Classification Pink rot on avocado fruits and stems is caused by Cephalothecium roseum Corda.

It is one of the Fungi

Imperfecti in the order Moniliales (Hyphomycetes), Family Moniliaceae, subfamily Hyalodidymae (Clements and Shear, 1931). Cephalothecium roseum Corda is considered by many authors to be synonymous with Trichothecium roseum Link. Among those who support this view are B. Peyronel, 1921; M. 0. Reinhardt, 1927; Gilman and Abbott, 1927; B. Oliveira, 1931; Charles Drechsler, 19*+3; and A. T. Henrici, 19^7. Generic Distinction P. A. Saccardo, 1886, separates Trichothecium Link from Cephalothecium Corda in the following manners Cephalothecium Corda.

Hyphae fertiles simplices,

longae, non nodosae, apice capitato-polysporae. Trichothecium Link.

Hyphae fertiles simplices, longae,

38 non nodasae, apice monosporae. Clements & Shear, 1931? use the following characteris­ tic to separate the two genera. Cephalothecium Corda. Trichothecium Link.

Conidia capitate Conidia solitary

Ernst Bessey, 1935, and Engler & Prantl, 1897, use this same distinction to distinguish between the two genera. According to Charles Drechsler, 19^3, in Trichothecium roseum Link, uniseptate, hyaline, or light colored conidia are borne singly on erect conidiophores; while in Cephalo­ thecium roseum Corda, such conidia are borne plurally in more or less capitate arrangement on erect conidiophores, simple, and septate.

"The distinction thus drawn has fallen somewhat

into disfavor from a belief that Trichothecium roseum Link and Cephalothecium roseum Corda, on which, respectively, the two genera would seem to have been founded, represent one and the same fungus.11 Validity of Names Drechsler, 19^3? questions the validity of Cephalo­ thecium because of its erection subsequent to Trichothecium. Link described Trichothecium roseum in 182^ (Species Plantarum, Link*s edition), and gave the following earlier references to the organism: erlang, p. 338*

Obs. I p . 16; Nees fung. p. **6; and Mart

39 Trichothecium roseum Link was previously called Trichoderma roseum (Pers. syn. p. 731), and Puccinia rosea Corda (Saccardo, IV, p. 173). The earliest reference for Cephalothecium roseum Corda given by Saccardo, 1886, in Sylloge fungorum hucusque cognitorum. is Corda, leones fungorum* V* 2, 1838, p. M-3* Conclusion If Trichothecium roseum Link and Cephalothecium roseum Corda are one and the same fungus, then the valid name depends upon the International Rules of Botanical Nomenclature. ever,

How­

if the two species belong to distinct genera, then

Trichothecium roseum Link is the name for the fungus which bears the conidia singly, and Cephalothecium roseum Corda is the name for the fungus which bears the conidia in capitate clusters. The fungus under investigation in this paper has been designated as C. roseum Corda, since the conidia are borne plurally in capitate clusters at the ends of erect, simple, conidiophores• According to M. 0. Reinhardt, 1927, “with poor nutri­ tion in T. roseum. the spore formation is reduced, producing the plant called C. roseum. with delicate spores, which have the surprising ability of germinating after being left for three years on agar.“

This statement only helps to increase

bO the problem of distinguishing between T. roseum Link and C. roseum Corda#

The statement would fit the situation

better if it were reversed. Morphology Cephalothecium roseum produces a prostrate, septate, hyaline mycelium which spreads over a substratum in a radial fashion.

The hyphae are freely branched and produce erect

conidiophores with capitate clusters of conidia borne at the tips.

The hyphae average 3.5 y. in diameter, and the conidio­

phores average 3*5 y» in diameter, and 188.50 y. in length. The conidia are hyaline, oblong-ovoid, uniseptate, and slightly constricted at the septum.

Engler & Prantl, 1897*

illustrate T. roseum with conidia not constricted at the septum, and C. roseum with conidia constricted at the septum. The conidia average 18.0 x 9*0 microns. The conidia are formed one at a time in basipetalous order.

The formation is unique in that each conidium appears

to be formed on the opposite side of the conidiophore from which the previous conidium was formed.

This formation is

illustrated in Figure I1*. Charles Drechsler, 193*+> describes Trichothecium Link as having species with discrete, constricting, stalked loops for capturing nematodes.

Hansen, 1938, describes organs of

capture among certain fungi which include most of the forms

C. ROSEUM: LIVING, SLIDE CULTURE X 4 4 .4

FIGURE 13 BEGINNING OF CONIDIAL CLUSTER AT TIP OF CONIDIOPHORE X £22.2

FIGURE IN­ FORMATION OF CONIDIA OF C. ROSEUM

FIGURE 15 ROSEUM: GERMINATION OF CONIDIA

Mf with 1-septate spores borne in a capitate or loosely capitate arrangement.

He states further that ,fit seems probable, simi­

lar solitary non-constricting loops and similar globose bodies will ultimately be found associated in other nema-capturing fungi, and undoubtedly in more than a few of the supposed saprophytes described from excrement of various animals or from decayed plant remains, that are compiled in the Sylloge Fungorum, in such genera as Cephalothecium Corda and Trichothecium Link,”

The author, however, has not observed any

nema-capturing organs in C. roseum. ENVIRONMENTAL RELATIONS OF C. ROSEUM The habit of growth and the vigor of C. roseum are regu­ lated by certain environmental influences which affect the degree of sporulation, the rate of vegetative growth, and the general appearance of the organism. These environmental influences include:

(1) the rela­

tive humidity of the atmosphere, (2) the light intensity, (3) the temperature, and (^) the substratum on which it is growing. The Relative Humidity of the Atmosphere The amount of available moisture present in the atmos­ phere has a direct effect upon the habit of growth of C. roseum. By regulating the relative humidity of closed chambers in which

^5 C . roseum was growing, the effects upon the degree of sporulation and the rate of vegetative growth were observed. Three experiments were conducted to determine the effects of different relative humidities upon the growth of C. roseum.

For the three experiments, the fungus was grown

in humidity-regulated chambers at a constant temperature (Methods, p. 12),

The substratum used for each experiment

was varied as follows: Experiment 1.

Uniform pieces of ripe avocado,

2,

Sabouraudfs maltose peptone agar,

3.

Sabouraud1s dextrose peptone agar.

C, roseum was allowed to develope in the regulated humidity chambers for seven days.

The results of these experi­

ments are presented in Tables VIII, IX, and X. It is concluded that when the relative humidity is between 70 and 100 per cent, at 20°C. - V 3, spopulation is heaviest.

As the relative humidity is decreased, the degree

of sporulation decreases accordingly, until a point where obvious indications of spore production stop. Between 50 and 70 per cent R,H., the vegetative growth seems to be at its maximum with a cottony white mycelium formed on the surface of the substratum.

The strands of

hyphae appear to lose their prostrate growing habit and become aerial in habit.

As the relative humidity is decreased, the

rate of vegetative growth becomes less, and the mycelium becomes a small closely compact mass of hyphae.

k6

TABLE VIII EFFECTS OF RELATIVE HUMIDITY UPON THE GROWTH OF CEPHALOTHECIUM ROSEUM SUBSTRATUM— PIECES OF AVOCADO

Relative Humidity at 20°C.

Incubation Period

Results

89.7

7 days

Prostrate, loose, webby, spreading growth. Heavy sporulation.

87.5

7 days

Prostrate, loose, webby, spreading growth. Less sporulation.

77.b

7 days

Growth slightly more com­ pact, less spreading. Slight sporulation at center of culture.

66.3

7 days

Heaviest vegetative growth. Cottony, aerial hyphae 3 mm above substratum. No visible sporulation.

5»t.6

7 days

Very compact, prostrate, not spreading. No visible sporulation. Reduced growth

b7

TABLE IX EFFECTS OF RELATIVE HUMIDITY UPON THE GROWTH OF CEPHALOTHECIUM ROSEUM SUBSTRATUM— SABOURAUD'S MALTOSE PEPTONE AGAR

Relative Humidity at 20°C.

Incubation Period

Results

♦Very meager growth, no sporu­ lation.

89.7

7 days

87.5

7 days

Prostrate, loose webby, spread­ ing growth. Heavy sporulation

77A

7 days

Prostrate, loose webby, spread­ ing growth. Slightly less sporulation.

66.3

7 days

Increased vegetative growth. Less sporulation.

5^.6

7 days

Loose, cottony, aerial hyphae. Reduced sporulation.

>+3.9

7 days

Reduced growth. Low, compact, not spreading. No visible sporulation.

3^.9

7 days

Cottony vegetative growth. Moderate sporulation.

Deviation from the apparent trend of growth and sporulation.

bS

TABLE X EFFECTS OF RELATIVE HUMIDITY UPON THE GROWTH OF CEPHALOTHECIUM ROSEUM SUBSTRATUM— SABOURAUD'S DEXTROSE PEPTONE AGAR

Relative Humidity at 20°C.

Incubation Period

Results

89.7

7 days

Prostrate, loose, webby, spread­ ing growth. Heavy sporulation.

87.5

7 days

Prostrate, loose, webby, spread­ ing growth. Less sporulation.

77A

7 days

66.3

7 days

Heavy vegetative growth. Less sporulation than at 87*5$.

5*f.6

7 days

Heavy vegetative growth, less sporulation.

^3.9

7 days

Reduced growth. Low. compact, not spreading. No visible sporulation.

3^.9

7 days

Prostrate, spreading growth. Some sporulation.

* lation.

*Very meager growth. lation.

No sporu­

Deviation from the apparent trend of growth and sporu­

*+9 After each experiment the growing cultures were all placed under a bell jar and kept at room temperature*

After

two days, all the cultures looked the same in degree of sporu­ lation and rate of vegetative growth*

This helps to illus­

trate the direct effects of the relative humidity upon the growth of C. roseum* The effects of relative humidity upon the prevalence of C* roseum on stems in avocado orchards.

Five adjoining

Fuerte avocado trees in a Yorba Linda orchard were found to have stems infected with C. roseum*

The fungus was also

found on fallen twigs in the litter beneath the trees.

The

abundance and habit of growth of C. roseum was observed on these trees for the period from September, 19*+8, to November, 19^9. During the hot summer, and dry fall months of the year, the growth of C. roseum on avocado stems is at a mini­ mum.

The fungus can be found in only one or two instances on

an infected tree, and there, in only the darkest and most pro­ tected portions.

C. roseum was found growing in stem scars

where the dead stem had not yet completely abscissed, and under layers of dead bark*

When the fungus is exposed to the

air it appears as small, compact, masses of conidia. color is dark pink to a light orange* hyphae present.

The

There are very few

When the organism is protected beneath dead

bark or in a stem scar, there are more hyphae present, but

50 less conidia and color* During the hot, dry period, C. roseum could not be found in the litter beneath the trees.

With the first damp

weather, the presence of C. roseum was detected.

During the

cold, wet period from December, 19^8, to February, 19^9, the fungus was extremely abundant in certain orchards, both on branches in the trees as well as in the litter beneath the trees. During this period the organism grows rapidly, becomes cottony in appearance, and takes on a bright pink hue.

It

grows abundantly in exposed locations and is very conspicuous. As the weather becomes drier in the Spring, the preva­ lence of the organism begins to decrease again.

Any sudden

increases in the relative humidity of the atmosphere, such as foggy weather or rain, brings about a corresponding increase in the prevalence of C. roseum. The Light Intensity Observations made in the field showed that C. roseum growing on Fuerte avocado stems could be found more abundantly in the darkest and most shaded portions of the trees.

Unpruned

Fuerte avocado trees form a very dense canopy down to the ground, and the heavy shade beneath these trees offers an excellent environment for the growth of C. roseum.

Branches

exposed to full insolation do not become infected with C. roseum.

51 To determine the effects of light intensity upon the rate of growth of C. roseum, four Petri dish cultures of C. roseum were exposed to a constant light intensity for five days at 75°F.

Four other cultures were kept in a dark chamber

for the same length of time and at the same temperature. (Methods, p. 16).

The results of this experiment are pre­

sented in Table XI. The cultures of C. roseum grown in the dark were nearly twice the size of those grown in constant light.

The degree

of sporulation appeared to be about the same in both cases. From the observations made in the field and in the laboratory it is concluded that light is an inhibiting factor upon the growth of C. roseum. Effect of Temperature The conidia of C. roseum will germinate at 7*5°C. on corn dextrose agar media.

Simonet, 1926, observed a minimum

of 8-10°C. for conidial germination. Oliveira, 1930, observed o vestiges of mycelial growth at 3-5 C., conidial germination at 5-7°C., and the formation of conidiophores after twelve days at 6-8°C., using sweet potato dextrose agar media.

The

optimum temperature for the growth of C. roseum is 20-22°C., and the apparent maximum is about 38°C. (Oliveira, 1930). Field observations in Fuerte avocado orchards during the period from September, 19^8, to November, 19*+9* showed

52

TABLE XI EFFECT OF CONSTANT LIGHT INTENSITY UPON THE GROWTH OF CEPHALQTHECIUM ROSEUM

Environment

Exposed to light. 230 ft. c. 75.1°F.

Absence of light 75. 2°F.

Petri Dish No.

Incubation Period

Diameter of Cultures in mm.

1

5 days

20.0

2

5 days

20.0

3

5 days

10.0

if

5 days

18.0

1

5 days

35.0

2

5 days

31.0

3

5 days

2if.6

if

5 days

36.0

Av. 17.0

. ~ g Av. 31*5

53 that C. roseum is more prevalent during the cold period of the year; and at a minimum during the warm season. The hot weather during the early part of September,

19*+8 , was the least favorable period observed for the growth of C. roseum. and the cold period during January, 19^9, was the most favorable period. The two growth-effecting factors, temperature and rela­ tive humidity, cannot be considered separately, and a consider ation of the effects of one must necessarily include the effects of the other. It is concluded that a cool and moist atmosphere is most favorable for the growth of G. roseum. and that a hot, dry atmosphere is least favorable. Effects of the Substratum It appears that the infection and subsequent growth of C. roseum on avocado fruits depends partly upon the degree of maturity and the ripeness of the fruit. To determine the effects of ripeness and degree of maturity of the fruit upon the growth of C. roseum. the following experiment was conducted. Harvested Fuerte avocados, from three chosen trees, were brought into the laboratory and were inoculated at different times from the beginning to the end of the picking season (November to June)•

It was observed that fruits

5^ picked early in the season did not become infected as easily or as quickly as those harvested in the middle of the picking season*

Fuertes harvested early did not become infected until

the fruit was soft ripe, but those harvested later in the season became infected within two or three days after picking if kept in a moist chamber.

Other factors, including changes

in temperature, are probably responsible for much of the difference which occurs in the rate of infection and degree of susceptibility between avocados. Susceptibility of different varieties.

The Nabal,

Anaheim, Hass, Fuerte, and Harmon varieties of avocados were inoculated with C. roseum to determine if there are any differ­ ences in the degrees of susceptibility to infection (Methods, p. 18).

The results of this experiment are presented in

Table XII. It is concluded that there is little if any differ­ ences in the degrees of susceptibility to infection by C. roseum among the five varieties of avocados tested.

It is

believed that any differences which do occur are due primarily to the degree of maturity and ripeness of the fruit, and to the factors which influence the growth of C. roseum. The relationship between C. roseum and the close proxim­ ity of avocado and citrus plantings.

Many citrus groves in

southern California, which are situated in districts favorable

55

TABLE XII SUSCEPTIBILITY OF FIVE DIFFERENT VARIETIES OF AVOCADOS TO INFECTION BY C. ROSEUM THREE FRUITS OF EACH VARIETY TESTED

Variety

Number of Fruit Infected

Number of Days before Fungus Atmeared on Each Fruit

1st fruit

2nd fruit

3rd fruit

Fuerte

3

5

7

12

Hass

3

10

6

13

Nabal

3

9

9

7

Anaheim

2

5

-

12

Harmon

2

18

6

-

56 for avocado production, are being interset with avocado trees. It is interesting to note that there are over fifty genera of fungi which occur on both citrus and avocados (Seymour, A.B., 1929). Two Valencia and two Navel oranges, two Lisbon and two Eureka lemons, and two grapefruits, were inoculated with a water suspension of conidia made from an isolate of C. roseum. through wounds made in the skin of the fruits. bated for several weeks in moist chambers. the fruit by £• roseum took place.

They were incu­

No infection of

C. roseum is inhibited by

citric acid (Verona, 1930). Culturing C. roseum in vitro.

For cultivating C. roseum

in the laboratory, Sabouraudfs dextrose peptone agar and corn dextrose agar proved to be very satisfactory.

Sabouraud’s

maltose peptone agar was not as satisfactory, although it did support a growth of C. roseum. Oliveira, 1930, obtained good results by using a sweet potato dextrose agar medium.

Le Clerg, 1930, and Oliveira,

1930, tested the reaction of C. roseum to many different artifi­ cial media and reference to their work will give an adequate list of media favorable to the growth of C. roseum.

The

results of Le Clerg*s work is reprinted in the Appendix, page 79.

57 CONTROL OF C . ROSEUM ON AVOCADO FR U ITS AND STEMS

From the results obtained during this investigation and the field observations made during several years of experience managing an avocado grove in Yorba Linda, methods of control to prevent the infection of avocado fruits and to reduce the prevalence of C. roseum in avocado orchards are suggested. Proper Harvesting of the Fruit Avocados should be clipped with just enough of the pedicel remaining intact to protect the stem end of the fruit from infection.

If too much of the pedicel is left attached

to the fruit, its sharp, clipped end may scar or wound other fruit.

If the pedicel is clipped too close to the surface,

the clippers may cut through the skin exposing a wound to infection by C. roseum.

Every precaution should be exercised

to prevent any wounding of the fruit. Time of Harvesting Avocados should not be picked while there is water hanging on the fruits and inthe

trees.

This water is an

excellent carrier for the transmission of fungous spores from one fruit to another inthe picking bags and field boxes. Very often, a wait of one or two hours in the morning is suffi­ cient to allow evaporation of the water from the trees.

58 Preventing Small Animal and Insect Damage Means to reduce or eliminate the population of any small animals such as chewing insects which would wound the fruit and expose them to infection should be employed. Insects or small animals carrying eonidia on their bodies would be a convenient means of infecting fruit. Pruning Trees Removing dead wood from avocado trees has two advan­ tages.

First, the dead twigs and branches are a favorable

substratum for the growth of C. roseum. and their removal reduces the prevalence of the fungus in the trees.

Secondly,

dead twigs and branches are responsible for much of the scarring and wounding of fruit during wind storms, which not only lowers the grade of the fruit, but which also offers avenues for infection by C. roseum. Selective pruning of branches to reduce excessive shading and to increase the light intensity beneath the trees will help to reduce the prevalence of C. roseum in avocado orchards.

This practice will also help to eliminate some of

the crowding and competition between neighboring trees. Excessive pruning of loiter branches to increase the ventilation and the penetration of light is not recommended. First, this is contrary to the habit of most avocado trees, and secondly, it will increase the loss of valuable moisture

59 from the ground. The Litter Beneath Avocado Trees Removal of the accumulation of leaves and small twigs from beneath avocado trees to reduce the prevalence of C. roseum is not recommended.

The litter is an invaluable

aid for the retention of soil moisture by protecting the ground surface from the effects of the sun and wind, and also by offering a continual source of humus for the soil.

How­

ever, large branch prunings should not be allowed to accumu­ late under trees.

They are of no value to the trees, and

offer a favorable substratum for the growth of C. roseum. Irrigation Methods Irrigating avocado orchards with ditches or low-head rotary sprinklers is most satisfactory. up into the trees using these methods.

Water is not sprayed Overhead sprinklers,

or sprinklers which throw the water upwards are not recommended. This excessive spraying into the trees only favors the spread of fungous diseases. Protection of Harvested Avocados Present day avocado packing companies protect their fruit from spoilage by storing the fruit in well ventilated storage chambers, maintained at lfO-^5°C.

The United Avocado

6o Grovers Association at La Habra, California, has not observed any fruit spoilage in their storage chambers. The present volume of avocado production in California is such that long storage periods for the fruit are not neces­ sary.

Eventually the volume of production will increase to

the point where fruit storage and preservation will present a greater problem; and then methods for preserving fruit harvested for longer periods will have to be improvised.

SUMMARY Cephalothecium roseum is a facultative saprophyte which occurs on avocado fruits and stems# C. roseum was isolated from spoiling, market fruits, and from infected avocado stems#

The morphological charac­

teristics used to identify C. roseum were observed by growing the fungus on slide cultures. Artificial inoculations of avocado fruits and stems were

made to establish the parasitism of C. roseum.

Sec­

tions of infected stem tissue were prepared to verify the parasitic nature of the fungus on avocado stems. The effects of environmental influences upon the growth and vigor of C. roseum were tested in the laboratory and in the field.

These environmental influences included

(1) the relative humidity of the atmosphere, (2) the light intensity, (3) the temperature, and (^) the substratum. The results of this investigation indicate that C. roseum is one of the most frequently occurring and most aggressive fungi on harvested avocados.

Artificial inocula­

tion tests demonstrated that the infection of healthy fruits is possible only through the exposed pedicel scar or through an open wound; and that infection cannot occur through an unbroken cuticle.

A high relative humidity and suitable

temperature are necessary before infection will occur. C. roseum will infect unharvested avocados providing that

61

62 there is a wound and sufficient moisture in the air to favor the growth of the organism. The infection of avocado fruits produces dark sunken areas on the surface of fruit and causes necrosis of the flesh.

Under moist conditions, the fungus develops small

compact masses of hyphae and conidia on the infected surfaces. C . roseum occurs as a saprophyte on dead avocado branches and twigs which persist in the trees and on those vihich are found in the litter beneath the trees. also occurs as a parasite on living stems.

The fungus

It is most commonly

found on small branches which are dying back from the tip, in cankerous lesions on green stems, in stem scars, or beneath the bark on larger limbs.

Sections of infected stem tissue

show that the fungus penetrates the living cells of the host. Attempts to demonstrate that C. roseum causes the die-back of small stems, or cankerous lesions, were not successful. A survey of avocado orchards in the Yorba Linda, Placentia, and La Habra areas of southern California indicate that C. roseum is distributed unevenly throughout Fuerte orchards, and that its frequency of occurrence on avocado stems is relatively small.

The fungus was found on Fuerte

stems, and not on stems of other varieties. Cephalothecium roseum Corda belongs to the Fungi Imperfecti and is considered by many authors to be synonymous with Trichothecium roseum Link.

The hyphae are hyaline,

63 septate, freely branched, and produce erect condiophores with capitate clusters of pear-shaped, two-celled conidia at the tips. C. roseum is commonly found in fruit and vegetable produce markets where it is responsible for the spoilage of many fruits and vegetables.

The importance of C. roseum is

indicated by the large number of hosts and substrata which favor the growth of the organism. The habit of growth and the vigor of C. roseum are regulated by environmental influences which affect the degree of sporulation and the rate of infection and growth.

A high

relative humidity accelerates the infection and subsequent growth of the fungus and increases the degree of sporulation. Strong or prolonged light intensity inhibits the growth of C. roseum.

The minimum temperature for growth is about 8°C.,

the optimum 20- 22°C., and the apparent maximum is about 38°C. The prevalence of C. roseum on avocado stems is at a maximum during the cool, wet period of the year; and at a minimum dur­ ing the warm, dry season. It appears that the infection and subsequent growth of C.

roseum on avocado fruits depends partly upon the degree

of maturity and the ripeness of the fruit, and that there is little if any differences in the degrees of susceptibility among the Nabal, Anaheim, Fuerte, Hass, and Harmon varieties of avocados.

Perhaps because the growth of the fungus is

6b inhibited by citric acid, C. roseum does not infect citrus fruits* Field observations and the results of this investiga­ tion indicate that the prevalence of C* roseum may be reduced by the application of certain cultural practices*

These

include proper picking and handling techniques to prevent wounding of the fruit; harvesting when there is no liquid water on the trees; removing dead wood which offers a suitable substratum for the fungus and which helps to scar the fruit; pruning out crowded branches to increase the light intensity and ventilation under trees; and employing irrigation methods which do not spray water up into the trees. Fruit spoilage may be prevented by storing avocados in well ventilated storage chambers, maintained at

2°C.

Means to prevent spoilage in the markets have not been worked out.

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APPENDIX

77 TABLE XIII FOUR SPOILAGE FUNGI IN TEE ORDER OF THEIR DECREASING AGGRESSIVENESS ON FUERTE AVOCADOS

Chamber Number 1 2 I 5 6 7 8 9 10 11 12 13 14 1! ?

1* C. roseum 1st 2nd 2nd 4th 2nd 2nd

2nd 1st 1st





1st

2nd 1st

-

-

3rd 2nd —



-



1st 3rd

4th

-



1st

-



-

-



1st 1st

-

-



-

-

-

1st 1st 2nd 4th 2nd 1st -

1st 3rd -

1st 3rd -

1st 1st 1st -

1st

1st

• •

2nd

-

-

-

-

-

2nd -



•»







4th 2nd

3rd



2nd





26







27

2nd 1st

1st 3rd

4th 4th

28

-



1st 3rd

17 18 19 20 21 22 2? 24 25

1st



-

-

16

2* 4* 3^ R. nigricans Cladosporium Acrostalagmus

_

2nd

1, Cephalothecium roseum: 2, Rhizopus nigricans ^*

1st to 4th decreasing aggressiveness. - Unaggressive or not present at all.

78

TABLE XIV MANNER IN WHICH C. ROSEOM INFECTS HEALTHY NABAL AVOCADOS

Conidial Suspension

No. of Incubation Fruit Chamber

No Infec­ tion

Infection

2

moist

X

2

open

X

2

moist

2

open

2

moist

X

2

open

X

On unbroken cuticle X

In X-cut wound X

In pedicel scar

79 TABLE XV

CULTURAL DATA FROM LE CLERG, 1930

Media upon which Trichothecium roseum was grown:

1*

Czapek

Cane Sugar, 30g$ NaNO^, 2g; lyiPO^, lg; MgS0if.7H20, 5g> KCi, 5g 5 FeSO^, .Olg; agaragar, l5g; 1 liter dist. water.

2.

Gelatin

I50g gelatin; water to make 1 liter.

3.

Potato

250g of raw ground potatoes; 0.5 liter water. Cook one hour and filtered; made up to 1 liter. 15g agar-agar added.

b.

Peptone

lOg bacto-peptone; I5g agar-agar; 1 liter water.

5.

Starch

20g starch; water to make 1 liter; 5g agaragar.

6•

Malt Extract

lOOg Puritan Pure malt extract; 1 liter water; l5g agar-agar.

7.

Wakesman Medium

Used to cultivate fungus on the five sugars maltose, dextrose, d-mannose, levulose, and inulin.

Formula used:

sugar lOg; peptone

5g; KgHPO^, lg; MgS0if.7H20, 0.5g; agar-agar, 15g; 1 liter water. (Continued on next page)

80 TABLE XV (Continued) CULTURAL DATA FROM LE CLERG, 1930

Cultural Characteristics of Trichothecium roseum after b-7 Days; 22-25°C. 1.

Czapek

Colonies somewhat spreading, white at first, later

turning pink, flat, round, floccose;

consisting almost entirely of conidiophores. Growth suppressed. abundant.

Spore production very

Reverse colorless to pale pink.

2.

Peptone

Same as Czapek.

3*

Starch

Same as Czapek.

Potato

Same as Czapek.

5.

Maltose

Same as Czapek.

6.

Inulin

Same as Czapek.

7.

Levulose

Same as Czapek.

8*

Dextrose

Same as Czapek.

9.

d-Marmose Same as Czapek, except that they are small and turn pink very much sooner.

10.

Malt Extract

Colonies same

as on Czapek, except that

growth is more rapid and spore production much more abundant. 11.

Gelatin

Same as on malt extract.

Liquefication.

81 TABLE XVI FUNGI WHICH INFECT AVOCADO FRUITS

Name

Pathogen

Rhizopus rot

Rhizoous nigricans

Dothiorella rot, or surface rot

Botryosohaeria ribis

Anthracnose rot Pink rot -

Horne, 193^

Colletotrichum gloeosoorioides Cephalothecium Alternaria

sdp.

sod.

Scab

CladosDorium

Eye rot

Fusarium

-

Reference

sdp.

sod.

Pestalozzia

sod.

ti

It

it

II

n

II

it

II

ti

It

it

tf

it

tl

Green mold of apples and pears

Penicillium exoansum

it

II

Blue mold of Citrus

Penicillium digitatum

it

II

Green mold of Citrus

Penicillium italicum

it

It

Brown rot fungus

Phytophthora citrophthora

n

It

Dry rot

Diplodia spp.

H

It

Green rot of apricots

Sclerotinia sclerotorium

II

It

Gray mold of stone fruits

Botrytis cinerea



II

(Continued on next page)

]

82

TABLE XVI (Continued) FUNGI WHICH INFECT AVOCADO FRUITS

Name

Pathogen

Reference Horne, 1926

Wine colored rot

Sclerotinia libertinia

Wine colored rot

Botrytis vulgaris

tt

tt

Gloeosnorium

ii

tt



s d d .

U n iv e rs ity o f S o u *1-'- n

o rn m ifflffiMrT