J. Phytopathology 118, 3—8 (1987)© 1987 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0031-9481

Horticultural Crops Quality Laboratory, USDA-ARS, Beltsville, Md., U.S.A.and Botany Department, University of Maryland, College Park, Md., U.S.A.

Influence of Temperature and Relative Humidityon Germinability of Mucor piriformis Spores

REGINA B . BERND, G . A . BEAN, W . S. CONWAY and H. E. MOLINE

Authors' addresses: REGINA B. BERND, National Center for Genetic Resources and Biotechnology,CENARGEN/EMBRAPA, P.O. Box 10.2372, 70.77D-Brasilia, DF (Brazil). G. A. BEAN, Depart-ment of Botany, University of Maryland, College Pk., MD 20742 (U.S.A.). W. S. CONWAY and H.E. MOLINE, United States Dept. of Agriculture, Agricultural Research Service, Horticultural Crops

Quality Laboratory, BARC-W, Beltsville, MD 20705 (U.S.A.).

Hth one figure

Received November 5, 1985; accepted March 17, 1986

Abstract

Studies were conducted to determine the influence of temperature and relative humidity (RH)on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °Cand their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1year at 0°C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH nolonger germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C,RH had little effect on spore germinability. The effect of temperature and RH on percentage sporegermination also varied. At all temperatures studied, spore viability decreased more rapidly with timeat 100% RH than at 75 or 90% RH. The least favorable temperature-humidity combination, 30 °Cand 100% RH, decreased spore germination from 100% to less than 1 % in 14 days.

Zusammenfassung

Einflufi von Temperatur und relativer Luftfeuchtigkeitauf die Keimfahigkeit von Mucor piriformis Sporen

Es wurden Untersuchungen durchgefiihrt, um den Einflufi von Temperatur und relativerLuftfeuchtigkeit (RH) auf die Keim- und Lebensfahigkeit von Mucor piriformis Sporen festzustellen.Die Sporen Iiberlebten eine Lagerung bei 35 °C nicht, und ihre Uberlebensrate verringerte sich sehrschnell bei 30 °C. Jedoch bei 0°C blieben die Sporen uber ein Jahr lebensfahig. Die Lebensfahigkeit derSporen wurde auch durch die RH signifikant beeinflufit. Sporen, die bei 26 °C und 100% RH gelagertwurden, keimten nach 35 Tagen nicht mehr, wahrend Sporen, die bei 75 oder 95% RH gelagert

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4 BERND, BEAN, CONWAY smd MOLINE

wurden, 65 Tage lang keimten. Bei 20 °C hatte die RH kaum Einflufi auf die Sporenkeimfahigkeit. DieAuswirkung von Temperatur und RH auf die prozentuale Sporenkeimung war auch unterschiedlich.Bei alien untersuchten Temperaturen verringerte sich die Sporenlebensfahigkeit schneller bei 100%RH als bei 75 oder 90 % RH. Bei der ungiinstigsten Temperatur-Liiftfeuchtigkeitskombination —30 °C und 100% RH — verringerte sich die Sporenkeimung von 100% zu weniger als 1 % innerhalb14 Tagen.

The importance of post-harvest diseases is illustrated by the heavy losses thatoccur between harvest and consumption of fruits and vegetables. A major causeof these losses is attributed to decomposition by microorganisms (ECKERT andSoMMER 1967, RYALL 1965). Mucor pirifonnis Fisher causes post-harvest decay ofnumerous fruits and vegetables, including strawberries, raspberries, blackberries,pears, apples, and stone fruits (BERTRAND and SAULI-CARTER 1980, DENNIS andMouNTFORD 1975, LoPATECKi and PETERS 1972, SMITH et al. 1979). During March1984, M. piriformis was responsible for the loss of 5,000 bushels of Deliciousapples at a single fruit packing company in Pennsylvania, representing a loss ofapproximately $30,000 (J. Rice, pers. comm.). Wounding of fresh fruits andvegetables during harvesting and handling cannot be completely avoided (ECKERT

1975, SoMMER 1982). Wounded produce is especially susceptible to infection bymicroorganisms that contaminate it in the packing house (BARKAI-GOLAN 1966,SMITH et al. 1971). Thus, inherent in the successful control of post-harvestpathogens such as M. piriformis are measures used to reduce the level of inocolumin storage facilities. Manipulation of temperature and relative humidity (RH) arethe most common physical factors recommended for control of post-harvestdecay (ECKERT 1977, RIPOON 1980, SPOTTS and PETERS 1982). Low temperaturestorage maintains the quality of many fruits and vegetables by retarding growthand sporulation and by suppressing metabolic activity of many fungi (DENNIS andBLIJHAM 1980, MATSUMOTO et al. 1969).

Mucor piriformis presents a unique problem because it causes decay at 0°Cand cannot be controlled by approved fungicide treatments. There are conflictingreports concerning the response of M. piriformis spores to temperature and RH(SNOW 1949, SPOTTS 1985, SPOTTS and PETERS 1982).

The objective of this study was to investigate the interaction of temperatureand RH on survival of M. piriformis spores under conditions similar to thosewhich might be encountered during the harvesting, storage, and marketing offresh produce.

Materials and Methods

The strain of M. piriformis (ATCC #38314) used in this study was obtained by single sporeisolation from peaches growing in Maryland. Cultures were stored in darkness at 0°C on Difco potatodextrose agar (PDA) slants. For spore production, M. piriformis was grown on PDA in darkness at20°C for 5 days in disposable Petri dishes (100 x 15 mm). Fungal spores were transferred from culturemedia to autoclaved gum tree {Nyssa syhatica Marsh) wood chips. Wood chips (8X15 mm) werecoated with spores by bringing them into contact with the surface of sporulating M. piriformiscolonies growing on PDA. The chips were then placed in sterile plastic Petri dishes (100 X 15 mm)without medium. Preliminary experiments showed that spores did not germinate on the wood chips

Influence of Temperature and Relative Humidity 5

and viability of spores was similar to that on glass surfaces. The dishes containing the spore ladenwood chips were placed in sealed chambers (Gaspak 100 System polycarbonate jars, 130D x230Hmm; Fisher Scientific, Silver Spring, MD), and RH's (40%, 75%, 90%, 100%) wereestablished according to the method of WEXLER and BROMBACHER (1952) and maintained bycirculating air through various proportions of glycerin-water mixtures or distilled water (100% RH)through the closed system which diaphragm pumps. RH's were monitored at least weekly using athermoelectric dew point hygrometer (EG & G Model 880, Waltham, MA). Temperatures of 0, 10,20,26, 30, and 35 °C were maintained by placing the RH chambers in incubators (Precision ScientificModel 818, Chicago, IL) set at the desired temperatures. Percentage spore germination was deter-mined at weekly intervals by removing wood chips from plates and placing them for 3 min in testtubes, each containing 1 ml sterile distilled water. After agitation, the spore suspension was pipettedfrom the tubes and evenly distributed over the surface of PDA plates. After an incubation period of18 h at 20°C in darkness, 100 spores selected at random on each plate were examined under lOOxmagnification and the percentage germination calculated. A spore was considered viable if a germ tubewas observed. Those plates with spores not germinating were held at 20^0 and re-checked daily for atleast 1 week. All experiments were replicated three times.

Results

The influence of temperature and RH on viability of spores of M. piriformisis summarized in Table 1. Both temperature and RH influenced the survival ofspores, although temperature had the greater effect. As the temperature ofincubation was increased, the survival rate of the spores decreased. Sporesincubated at 0°C germinated more than 1 year later, while those incubated at35 °C did not germinate after 24 h. At each temperature between 10 and 30 °Cspores held at the lowest humidity (40%) survived for the longest time. If weexamine the percentage spore germination (Fig. 1) we can see that the rate ofgermination dropped most rapidly at 100% RH. Although a few spores con-tinued to germinate after 35 days at 20°C and after 14 days at 26 and 30°C, thespore survival rate was significantly reduced at 100 % RH at these three tempera-tures compared with 75 and 90% RH. The few spores that remained viablecontinued to germinate for up to 77 days at 20 °C, for 35 days at 26°C, and for 28days at 30°C (Table 1).

Table 1Effects of temperature and relative humidity on the germinability of Mucor piriformis spores

% Relativehumidity

407590

100

0

365 + 3}''365 -1- a365 -t- a365 -f- a

10

154 a119 b112 b105 b

Incubation20

98 a77 b77 b77 b

temperature (°C)26

84 a63 b63 b35 c

30

49 a35 b35 b28 b

35

———

Mean number of days after which no spores germinated; spores stored at 0°C continued togerminate at termination of experiment at 1 year and those stored at 35°C did not germinate after24 h.Numbers followed by the same letter within each column did not differ significantly (P = 0.05)according to the LSD test.

M 21 28 3S

Tim* »t Cxpeaura Cdays)

42

108-

88^

oao

1ua.

14 2t 26 35

Tim* of Expocur* (days)

49

0o

a.20-

42 49

Tint* of Cdoya)

Fig. 1. The influence of temperature and relative humidity on percentage spore germination of Mucorpiriformis at various time intervals. Temperatures include A) 20 °C, B) 26 °C, and C) 30 °C. Relative

humidities at each of the temperatures are 75% (•) , 90% (A), and 100% (•)

Influence of Temperature and Relative Humidity 7

Discussion

Temperature and RH influence both the percentage germination ofM. piriformis spores and the long term viability. A combination of factors may beresponsible for the rapid loss in viability of M. piriformis spores when exposed tohigh temperature and RH. During germination, spores are more sensitive toextreme temperatures and lose viability and virulence, especially during pro-longed periods of exposure to high or low temperatures (DENNIS and BLIJHAM

1980, SMITH et al. 1979). At a high RH, spore germination is stimulated (PAGEetal. 1947); however, the germinating spores are more susceptible to high tempera-tures and the metabolic steps involved in spore germination may be retarded ortotally inhibited. Previous studies (ECKERT and SOMMER 1967) have shown thatenzymes are more readily inactivated when hydrated, slowing down or inhibitingmetabolic processes which occur during spore germination. High RH's stimulatespore germination; however, high temperatures may also be inhibitory to enzy-matic processes that are occurring in spores causing germination to cease. Freewater inside spores is normally in equilibrium with water surrounding the spore.Thus, spores held at a high RH would have a greater proportion of water in thefree state and less food reserves available in the protoplasm. Therefore, the freewater inside the spores, coupled with a high temperature, increases the metabolicrates and reduces food reserves in the spores for germination which could result ina rapid loss in viability of the spores (MEREK and FERGUS 1954). Two physicalproperties may also contribute to the lethal effect of the 30 °C and 100% RHtreatment. Heat is transferred more readily in wet air (ECKERT and SOMMER 1967),and high relative humidity enables a higher temperature to be reached in spores ina shorter period of time (SMITH and WORTHINGTON 1965).

Mucor piriformis spores fail to germinate at temperatures above 27 °C (SMITH

et al. 1979); however, they can survive at temperatures of 30°C (Table 1).Increasing the RH drastically reduces spore survival at higher temperatures. Sincethe pathogen cannot be effectively eliminated with approved fungicides, a combi-nation of high temperature and high humidity may place enough stress on thepathogen to reduce its potential pathogenicity. Sanitary practices such as steamcleaning or solarization of storage facilities and field boxes should remove residualinoculum that might cause decay of injured fruit. By understanding how environ-mental conditions affect spore survival and manipulating the environment to ourmaximum benefit we may place enough stress on the fungus to minimize itsimpact as a post-harvest pathogen.

This work was part of a thesis submitted by R. B. BERNfD to the Botany Department, Universityof Maryland, College Park, MD for the M.S. degree.

Use of a company or product name by the U.S. Department of Agriculture does not implyapproval or recommendation of the product to the exclusion of others which may also be suitable.

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