Induced changes in hyphal growth of Pilaira anomala

Induced changes in hyphal growth of Pilaira anomala

[ 53 1 Trans. Br . my col. Soc. 78 (3) 531-574 (1982) ] Printed in Great Britain NOTES AND BRIEF ARTICLES INDUCED CHANGES IN HYPHAL GROWTH OF PILAI...

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[ 53 1 Trans. Br . my col. Soc. 78 (3) 531-574 (1982)


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Nutritional requirements of Pilaira anomala converted to hyphal length per tip and are (Cesati) Schroeter were discussed in Fletcher pres ented with the yields in Table 1. As expected, there was a decline in yield with (1971). For some studies a maltose-peptone agar med ium has been found to be adequate, even at a dilution of the nutrients with differences in yield concentration of one fifth normal (MPA / s). between MP and MP 15being statistically significant Cultures grown in broth based solely on this or (P = < 0'001). In MP a filamentous mycelium lower concentrations, however, produced abnormal consisting of radiating branched hyphae was hyphal tips. This observation, previously unpub- formed. This contrasted with the crusty mycelium lished, was also made on other media oflow nutrient which developed in the less concentrated media. Measurements of the hyphal branching pattern status. In the present investigation dry weight yields and (H BP) of main hyphae i.e. the mean length of hypha form of hyphal branching pattern have been per tip showed a decline in this value with the measured on different concentrations of nutrient dilution of the components of the medium. The and observed under different physical conditions. differences between the three values of HBP for The medium used throughout contained maltose each isolate were statistically significant (between (40 gil) and mycological peptone (10 gil) [MP- MP Is and MP 110 for H-32, P = < 0'01; between broth; MPA-agar] or at lower concentrations: other pairs P = < 0'001). The crust is therefore the MP /s ; MPA /s and MP/lo. The isolate IMI macroscopic appearance of many closely packed 109387 (ex CMI, Kew) was used for the measure- hyphal (' mamillate') tips. Mamillate tips were also ment of dry weight and hyphal branching pattern, formed on MP (and in lower concentrations) at 37°, whereas the isolate H-32 (ex CBS, Baarn) was used the HBP values were lower than at 25° as were the for the measurement of hyphal branching pattern yields. Hyphal form has been recorded (but not only. illustrated) by phase-contrast microscopy and conFor dry weight measurement each 100 ml flask firmed by scanning electron microscopy . Figure lA contained 25 ml medium giving a surface to volume shows normal monopodial growth of IMI 109387 ratio of 1'0 approx . Four replicates were set up , on MP, the mamillate hyphal tips produced by each inoculated with a plug of mycelium and this isolate in MP Is (F ig. 1B), an increased numincubated at 25°C for 66 h. Measurements of ber of such tips in MP 110 (Fig. 1 C) and the hyphal branching pattern were made on the abnormal form in MP 110 of the isolate H-32 (Fig. mycelium grown on a broth culture, from an 1D ). The corresponding HBP values are> 140, c. inoculum of spores, over a period of 6 days (IM I 30, c. lS and c. 15/lm. The mamillate tip is a swollen structure approx. 109387) or 11 days (CBS H-32 ) at 25°. Counts were made of the number of tips on the 120 or 140/lm 12-24 /lm across, often wider than the hypha from distal portion of Somain hyphae . These values were which it arises. The diameter of the mamillate tips Table 1. Dry weight and hyphal branching pattern (H B P) in Pilaira anomala (means ± S.E.) Isolate IMI 109387

Isolate CBS H-32 HBP Gum)

Dry w t , Medium MP MP /5 MP/10

(mgyioo ml)

HBP (,urn)

141 ± 4' 5 16 ±5 '1 7

115"3±5"2 21'4 ±O '7 17'6 ±o'6

86'5 ±4'5 26'8±1 '1 21'O±l '2

HBP , length of main hypha /tip.

Notes and brief art icles


A. B.C

l Ofl um

F ig. 1. H yph al form in Pi/aira anomala. IMI 1°938 7 : (A) No rma l grow th on MP. (B) M amill ate tips on MP/ 5· (C) Mamillate tips on M P / 10. CBS H -3 2: (D ) M am illate tip s on MP / 10. (Cytoplasmic conte nts are not shown .)

increases with decrea se in HBP value as shown in Fig . 1. This swelling would appear to be associated with a weakness in the hyphaI wall as in the scanning electron micr ographs tearing occurred in the preparation. The surface to volum e ratio (SjV) of the medium in the 100 ml flasks used for dry weight yield s was 1 ' 0 approximatel y. When pieces of mycelium from MP j 5 were transferred to MPA j 5 in Petri dishes, the form of newly-emerged hyph ae was normal. In other experiments where cultures on M P j 5 were set up in Perri dishes (Sj V = 4'0) or in 1 I Roux bottles (SjV = 8'0 ) th e form of th e hyphal tip s was also normal. Lowerin g th e Sj V to 0'3 also reduced yield by approx. 50 % on MP , An att emp t was made to induce a yeast-lik e for m Trans. Br . mycol. S oc. 78, (3) (1982)

by lowering the oxidation potent ial by the incorporation of cysteine hydrochloride into the medium (Rippon, 1980). This was carried out in sloped gradient plates. Pilaira howe ver , was not able to tolerate the presence of cysteine in th e Petri dish, possibly due to some toxic volatile by-product of th is amino acid . Trinci (1974) in defining th e hyphal growth unit, a value related to the hyphal branching pattern here, suggested that branch initiation may be regulat ed by change s in cytoplasmic volume accompanying growth, in th at when the mean volume of cytoplasm exceeds a critical volume a new bran ch is initiated. Here, thi s hypothesis appli es in that in normal monop od ial hyphae ' apical dominance ' operates over a certain lengt h

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Notes and brief articles (volume) of hypha . In the mamillate form, branching has occurred at a much lower critical volume. These changes would indicate a breakdown in the integration of hyphae probably brought about by poor aeration ofthe medium, as they have primarily occurred at S /V ratios < 1'0 but not at > 4'0 (growth at a higher temperature may also be affecting similar processes). Thus abnormal growth may be due to fundamental changes in wall synthesis as suggested by Jones & Bu'Lock (1977). The latter workers induced effects similar to those obtained with Pilaira, by use of cyclic AMP. A feature of the morphological changes in Mucor spp ., however, was the production of septa. Septa were also formed in the delimitation of arthrospores in Mucor rouxii (Calmett e) Wehmer (Bartnicki-Garcia & Nickerson, 1962) but were absent from all forms of Pilaira. Whether these results have merely demonstrated another example of the plasticity of the mycelium of this fungus in response to the altered external


environment (F letcher, 1971) or whether altered forms of branching perform a more adaptive role will require further study. The authors wish to thank Mrs Dee Barrett for technical assistance. REFERENCES

BARTNICKI-GARCIA, S . & NICKERSON, W .] . (1962). Induction of yeast-like development in Mucor by carbon dio xide . Journal of Bacter iology 84, 829--840. FLETCHER, H.] . (1971). Some aspects of the biology of Pilaira anomala - an extremely versatile fungus . Journal of B iological Education 5, 229--237. JONES, B. E. & BU'LOCK, ] . D . (1977). The effects of

N ' , 0 " -dibutyryl adenosine-j ',5' -cyclic monophosphate on morphogenesis in Mucorales. Journal of General Microbiology 103, 29--36. RIPPON,]. W. (1980). Dimorphism in pathogenic fungi. Critical Reviews in Microbiology 8, 49--97. TRINCI, A. P. ]. (1974). A study of the kinetics of hyphal extension and branch initiation of fungal mycelia. J ournal of General Microbiology 81, 225-236.



School of Biological Sciences, Univ ersity of Ea st Anglia, Norwich NR4 7TJ

The polyacetylenic antifungal compound falcarindiol, isolated from carrot roots (G arrod, Lewis & Coxon, 1978), appears to have a broad spectrum of antifungal activity at low concentrations when assessed by measuring its effect upon spore germination (Garrod et al ., 1978 ; Kemp, 1978) and acts on the plasma membrane or on some process necessary for membrane function (Garrod, Lea & Lewis, 1979). The ED so for inhibition of germination of chlamydospores of Mycocentrospora acerina (Hartig) Deighton for this compound was 31'8,ug/ml (Garrod et al., 1978). M. acerina, the causal agent of liquorice rot, is one of the most important long-term storage diseases of carrots and celery (Rader, 1952; Mukula, 1957; Arsvoll, 1969; Derbyshire & Crisp, 1971 ; Day, Lewis & Martin, 1972). This investigation studies the effect of falcarindiol on hyphal growth of this pathogen and considers whether levels found in tissue are likely to be sufficient to account for the limitation of lesion spread. A series of 2'0 % ethanol solutions containing a range of falcarindiol concentrations were prepared; a solution containing 2'0 % ethanol only served as a control. Twelve small rectangular pieces of agar, Trans . Br . mycol. Soc. 78 , (3) (1982)

approximately 20 X 5 x 3 mm (thick), were cut from the edge of a 6-day-old colony of M. aeeTina on V8 juice agar (G arrod et al., 1978) grown at 15 ± 1 "C. Each block of agar bore a hypha I front approximately 10 mm from one edge . These blocks were placed on clean glass slides and surrounded by a carefully cut 'wall' of damp glass fibre paper, to support a large cover slip and to help maintain a high humidity (F ig. 1). Each of these systems was located on a Watson Microsystem 70 microscope, in a room at 15 ± 10, and the length of a single hypha on each slide was measured at 10 min intervals. When a constant rate of increase in hyphallength had been recorded (after about 40 min) 10,u1 of each solution was dropped, with the aid of a Hamilton syringe, on to the hypha 1tip under observation. The hyphal growth measurements were continued for a period of about 1 h. Prior to each measurement the cover slip was removed carefully and replaced immediately afterwards to avoid excessive loss of moisture. Microscope lamps were also turned off between readings to avoid heating effects on the hypha under observation and, at approximately 30 min intervals, 2-3 drops of water were added to the glass-fibre

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