Characterization of chlorate-resistant sectors from isolates of Fusarium moniliforme and F. proliferatum

Journal of Microbiological Methods 31 (1998) 175–184

Journal of Microbiological Methods

Characterization of chlorate-resistant sectors from isolates of Fusarium moniliforme and F. proliferatum Giovanni Vannacci*, Caterina Cristani Dipartimento C.D.S.L. Sezione Patologia Vegetale, Universita` degli Studi di Pisa, Via del Borghetto 80, 56124 Pisa, Italy Accepted 29 October 1997

Abstract Fusarium moniliforme and Fusarium proliferatum produce spontaneous chlorate-resistant sectors on chlorate-containing media. Most of these sectors are nitrate non-utilizing mutants (nit mutants). We recovered nit mutants as spontaneous chlorate-resistant sectors from 18 isolates of F. moniliforme, and 16 isolates of F. proliferatum of Italian origin. Three media, each containing two different chlorate concentrations, were used. The nit mutants were characterized by their colony morphology on different nitrogen sources. In some cases nitrate reductase activity was evaluated by testing for nitrite excretion. We found three classes of mutants (nit1, nit3, NitM) corresponding to those assigned in previous studies to mutants of Fusarium species, and, with a low frequency, two additional unexpected classes. One of these, designated Nit8, presumably reflected a mutation at a nitrite reductase structural locus as mutants were positive in the nitrite excretion test. Mutants that grew prototrophically only when reduced sulphur sources were added to the medium were assigned to the other class, and considered as sulphate non-utilizing mutants (sul). The majority of sul mutants responded to thiosulphate supplementation but two required cysteine or methionine. Complementation was also observed when some of these mutants where co-cultured on MM. The pattern of positive reaction obtained suggested that our sul mutants could result from mutations of at least three loci associated with sulphate metabolism. The sul mutants were recovered from F. moniliforme isolates only. Sectoring on chlorate-containing media provides a method for the isolation of Nit8 mutants from F. moniliforme and F. proliferatum, and of sul mutants from F. moniliforme.  1998 Elsevier Science B.V. Keywords: Chlorate resistant; Fusarium moniliforme; Fusarium proliferatum; Nitrate; Nitrite; Sulphate

1. Introduction Fusarium moniliforme (Sheld.) emend. Snyd. and Hans. and F. proliferatum (T. Matsushima) Nirenberg are members of the section Liseola, and until recently they were taxonomically synonymous. F. moniliforme is pathogenic to a variety of economically important crops. F. proliferatum causes a disease of asparagus (Asparagus officinalis L.) *Corresponding author. [email protected]

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known as Fusarium crown and root rot, but it is also frequently involved, together with F. moniliforme, in maize (Zea mays L.) ear and stalk rot [1–3]. Fusaria are considered to be genetically unstable because they easily produce sectors in culture that differ from the original colony in many characters, such as morphology, virulence or resistance to toxic compounds and this instability seems, in some cases, to be under genetic control [4,5]. Positive selection systems for isolation of suitable mutants, based on spontaneous resistance to toxicants, have been used with several fungal species for

0167-7012 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0167-7012( 97 )00101-2

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genetic investigation or to assess the diversity of compatibility relationships. Mutations for resistance to a particular drug (chlorate, selenate, p-fluorophenylalanine) are often associated with an auxotrophic requirement or non-utilization phenotype [6–9]. F. moniliforme and F. proliferatum produce spontaneous chlorate-resistant sectors on media containing chlorate. Most of these sectors are nitrate non-utilizing mutants (nit) with lesions in loci of the nitrate reduction pathway [2,5]. The nitrate assimilation pathway has been well characterized in both Neurospora crassa and Aspergillus nidulans. Mutants of these species are available which define the enzymatic steps of this pathway, including those mutated in a major regulatory locus (nit2, areA), responsible for nitrogen metabolite repression of nitrate assimilation and other pathways of nitrogen acquisition [6,10,11]. The nit loci identified in F. moniliforme are similar in number and phenotype to genes identified in N. crassa and A. nidulans. The nit mutants were found in F. moniliforme as chlorateresistant sectors, with lesions in seven different loci: a structural gene for nitrate reductase, a pathwayspecific regulatory gene and five genes controlling the production of a molybdenum-containing cofactor, which is necessary for nitrate reductase activity. In F. proliferatum the same classes of nit mutants are known as those recovered in F. moniliforme. No mutations affecting nitrite reductase, or the major regulatory locus, have been reported to date in these two species [2,5]. The nit mutants have often been used to force heterokaryons, and thereby to define vegetative compatibility relationships in these fungi. More information is available on nit mutant production in F. moniliforme than in F. proliferatum. In a preliminary investigation [12], we tested two groups of Italian isolates of F. moniliforme and F. proliferatum on chlorate-containing media to examine the production of nitrate non-utilizing mutants. Some of the sectors recovered from F. moniliforme were unable to grow well on any of the five nitrogen sources (nitrate, nitrite, hypoxanthine, ammonium and uric acid) employed to characterize the nit mutant phenotypes. These sectors were initially assumed to be similar to regulatory mutants such as nnu of Gibberella zeae [13] and nit2 of

Neurospora crassa, [14]. This preliminary diagnosis was later found to be incorrect, as the sectors proved to be sulphate non-utilizing mutants (unpublished results). Selection for resistance to selenate provides a system for the isolation of spontaneous sulphate non-utilizing mutants from fungal strains. Such mutants have been used to test for vegetative compatibility, with nitrate non-utilizing mutants, in F. oxysporum, F. oxysporum f.sp. melonis, F. moniliforme and Pseudocercosporella herpotrichoides [9,15,16]. No information is yet available concerning their production on chlorate-containing media. Within the framework of a vegetative compatibility group composition study of Italian F. moniliforme and F. proliferatum populations, the present work was undertaken to compare the frequency and physiological phenotype of nit mutants recovered from Italian F. moniliforme and F. proliferatum isolates and to confirm the production of sulphate non-utilizing mutants from chlorate-resistant sectors.

2. Materials and methods

2.1. Fungal isolates Eighteen isolates of F. moniliforme and 16 isolates of F. proliferatum obtained from commercial seeds and symptomatic plants of Italian origin (Table 1) were examined. Species were classified according to Nelson et al. [17] on the basis of fructification morphology on CLA, moreover F. moniliforme produces monophialides only, while F. proliferatum produces both mono and polyphialides.

2.2. Culture media PDA, Potato Dextrose Agar (Difco, Detroit, USA); CLA, Carnation Leaf Agar [17]; Basal Medium (BM) [18]: 1 l of distilled water, 30 g sucrose, 1 g KH 2 PO 4 , 0.5 g MgSO 4 .7H 2 O, 0.5 g KCl, 10 mg FeSO 4 .7H 2 O, 20 g agar (Difco), 0.2 ml trace element solution. The trace element solution was prepared by adding the following to 95 ml of distilled water: 5 g citric acid, 5 g ZnSO 4 .7H 2 O, 1 g Fe(NH 4 ) 2 (SO 4 ) 2 .6H 2 O, 0.25 g CuSO 4 .5H 2 O, 50 mg

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Table 1 Isolates of F. moniliforme and F. proliferatum used in this study

2.4. Media for phenotypic characterization

F. moniliforme

F. proliferatum

Isolate

Source of isolation

Isolate

Source of isolation

C142 / 86 C266 / 86 L1 / 83 Ms2 Ms4 Ms5 Ms6 Ms7 Ms8 Ms9 Ms10 Ms11 Ms12 Ms13 Ms14 Ms15 R175 / 87 R176 / 87

Onion Onion Flax Maize Maize Maize Maize Maize Maize Maize Maize Maize Maize Maize Maize Maize Salsola kali Salsola kali

A4 A6 A8 C212 C237 C109 / 86 C123 / 86 C219 / 86 C223 / 86 C262 / 86 Ga63 / 83 Ms3 Ms66 M1H M1E M3E

Asparagus spp. Asparagus spp. Asparagus spp. Onion Onion Onion Onion Onion Onion Onion Carnation Maize Maize Melon Melon Melon

Mutants were assigned to different phenotypic classes on the basis of their growth on BM containing different nitrogen sources: (1) 2 g l 21 NaNO 3 ; (2) 0.5 g l 21 NaNO 2 ; (3) 0.2 g l 21 hypoxanthine; (4) 1 g l 21 ammonium tartrate; (5) 0.2 g l 21 uric acid. Mutants showing a starved growth pattern on these media were incubated on different substrates prepared by using BM amended with 19.2 mM N equivalent of L-asparagine, L-cysteine, Lglutamine, L-methionine, L-proline, putrescine, taurine, urea. A MM in which sulphate (MgSO 4 .7H 2 O) was replaced by 0.5 g thiosulphate (Na 2 S 2 O 3 .5H 2 O) was also utilized [5,8,13,16]. Most mutants unable to utilize either nitrate or nitrite were separated into two different classes on the basis of nitrite excretion. Mutants were grown on urea medium (0.4 g l 21 urea added to BM) in a 9-cm petri dish for 4 days at 258C. The plates were flooded with a 3 M NaNO 3 solution (10 ml per plate). After 24 h, the nitrate solution was poured off and the presence of nitrite was revealed by adding the following to each plate: 1 ml of a sulfanilamide solution (75 ml distilled H 2 O, 25 ml concentrated HCl, 1 g sulfanilamide) and 1 ml of a color indicator (100 ml distilled H 2 O, 20 mg N-1-naphthyl-ethylenediamine.2HCl). Presence of nitrite was indicated by a bright purple colouring [6,18]. Positive tests were repeated at least twice. Phenotypic classes to which nit mutants were assigned are summarized in Table 2. Mutants showing sparse growth and no aerial mycelium on nitrogen-supplemented media but growing prototrophically on media supplemented with thiosulphate, cysteine or methionine, were considered sulphate non-utilizing (sul) mutants with mutations of some loci of the sulphur metabolic pathway. The sul mutants were tested for selenate resistance on MM without MgSO 4 .7H 2 O, amended with taurine (0.1 g l 21 ) and sodium selenate (1.0 g l 21 ) [15].

MnSO 4 .H 2 O, 50 mg H 3 BO 3 , 50 mg NaMoO 4 .2H 2 O; Minimal Medium (MM) [18]: 2 g of NaNO 3 added to 1 l of BM.

2.3. Media for nit production PDCL: 1 l of distilled water, 24 g Potato Dextrose Broth (Difco), 20 g agar, 20 g (PDCL2%) or 40 g (PDCL4%) KClO 3 ; MMCLA: 1 l BM, 2 g NaNO 3 , 1.6 g L-asparagine, 20 g agar, 15 g KClO 3 ; MMCL: 1 l BM, 2 g NaNO 3 , 20 g agar, 15 g (MMCL1.5%) or 40 g (MMCL4%) KClO 3 . The nit mutants were generated by transferring 6-mm mycelial disks from each isolate to the centre of a 9-cm diameter petri dish (at least 10 disks for each isolate and for each substrate) filled with 15 ml of the KClO 3 -containing media. Plates were incubated for 7–15 days at 258C with a 12 h dark / 12 h light cycle. Mycelial disks initially gave rise to colonies with restricted growth. Resistant sectors showing a faster growth rate than the remaining colony were transferred to MM and those that grew as thin expansive colonies with no aerial mycelium were considered nit mutants.

2.5. Complementation tests By pairing complementary nit mutants on MM the development of a dense heterokaryotic mycelium along the line of contact can be observed. Mycelial

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Table 2 Identifcation of nitrate non-utilizing (nit) mutants of F. moniliforme and F. proliferatum according to growth on different nitrogen sources [18] Mutant type a

Wt nit1 nit3 d Nit8 d NitM

Growth on nitrogen sources b

Nitrite excretion c

A

B

C

D

E

1 2 2 2 2

1 1 2 2 1

1 1 1 1 1

1 1 1 1 2

1 1 1 1 1

Slight Not tested No Yes Not tested

a

Mutant designation: nit1 (nitrate reductase structural locus); nit3 (pathway-specifc regulatory locus); Nit8 phenotype like nit6 mutants of Neurospora crassa [10] (nitrite reductase structural locus); NitM (molybdenum cofactor loci); Wt (wild type). b Growth on basal medium amended with different nitrogen sources: 1, typical wild-type growth; 2, thin growth with no aerial mycelium; A, NaNO 3 ; B, NaNO 2 ; C, ammonium tartrate; D, hypoxanthine; E, uric acid. c Yes: positive (bright purple) reaction; slight: some (light purple) reaction; no: negative (no purple) reaction. d Mutants unable to utilize nitrate and nitrite were classifed as Nit3 / 8. Most of them were subsequently tested for nitrite excretion.

disks (6 mm diameter) were placed 2 cm apart on MM and were incubated for 15 days at 258C. All nit and sul mutants recovered from the same parent were paired with at least one nit1, one Nit3 / 8 and one NitM mutant from the given parent. Also, seven sul mutants from different parents were paired in all possible combinations.

2.6. Genetic terminology The genetic terminology used in this study is in accordance with published information and the suggestions of Yoder et al. [19].

3. Results

3.1. nit mutant isolation Isolates of F. moniliforme and F. proliferatum produced spontaneous resistant sectors with a varying frequency on media containing potassium chlorate, depending on the isolate and medium used. Resistant sectors from both fungi were recovered from PDCL, above all when containing 20 g l 2 1 chlorate, with a higher mean frequency (sectors per colony) than on MMCL and MMCLA. On the other hand, the highest percentages, 77.2% for F. moniliforme and 79.8% for F. proliferatum, of sectors shown to be nit mutants was observed on MMCL4%. F. proliferatum, unlike F. moniliforme, did not produce sectors on MMCLA (Table 3). In all

cases, elevated variability was observed, due to the different ability of isolates of both species to produce nit mutants.

3.2. nit mutant phenotype identification Mutant phenotypes were assigned to the classes nit1, Nit3 / 8, NitM, according to the colony morphology on media containing different nitrogen sources (Table 2). Mutants that did not show normal growth on any of the nitrogen media used to characterize nit mutants were evaluated on other nitrogen and sulphur sources. As they grew with a prototrophic morphology only when thiosulphate, methionine or cysteine was added to the medium, they were considered sul mutants (Table 4). For both species, the relative percentage of mutants belonging to the different phenotypic classes was influenced by the medium. In F. moniliforme, roughly half of the nit mutants recovered were nit1 with all media used. NitM mutants were recovered with higher frequency from MMCL4% (30.2%) and MMCL1.5% (24.1%) while none was found from MMCLA. The number of Nit3 / 8 was highest from MMCLA, where they represented about half of the nit mutants recovered. On PDCL and MMCL the number of Nit3 / 8 decreased with increasing chlorate concentration. A small number of sulphate nonutilizing mutants were produced on PDCL2% by seven isolates (Table 3). The sul mutants recovered showed two growth patterns on sulphur sources, as two of them were unable to utilize thiosulphate.

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Table 3 Cumulative data of frequency and phenotype of nitrate (nit) and sulphate (sul) non-utilizing mutants of F. moniliforme and F. proliferatum from five media containing chlorate Medium

Isolates a

Sectors per colony b

Mutants % c

Tested d

Min.

Mean

Max.

Min.

Mean

Max.

Fusarium moniliforme PDCL2% 11 MMCL1.5% 11 MMCLA1.5% 11 PDCL4% 18 (11 f ) MMCL4% 18 (11)

0.6 0.0 0.0 1.0 (1.0) 0.6 (0.6)

2.1 0.8 0.4 1.8 (1.6) 1.0 (1.0)

3.2 1.2 1.6 2.4 (2.1) 1.3 (1.3)

4.8 0.0 0.0 20.8 (30.0) 46.2 (46.2)

18.4 56.3 54.8 53.7 (57.9) 77.2 (80.4)

33.3 91.7 100.0 81.3 (81.3) 94.4 (94.4)

Fusarium proliferatum PDCL2% 9 MMCL1.5% 9 MMCLA1.5% 9 PDCL4% 16 (9) MMCL4% 16 (9)

0.9 0.0 0.0 1.2 (1.2) 0.8 (0.8)

2.0 0.2 0.0 1.9 (1.9) 1.0 (1.0)

3.0 1.0 0.0 2.7 (2.7) 1.4 (1.4)

52.9 20.0 — 43.8 (43.8) 65.5 (67.5)

72.4 56.7 — 69.8 (73.0) 79.8 (79.4)

83.8 100.0 — 95.2 (95.2) 92.8 (86.0)

Mutant classes e nit1 %

NitM%

Nit3/8%

sul%

64 63 24 215 (113) 361 (215)

46.9 55.6 54.2 64.2 (60.2) 64.5 (63.3)

4.7 30.2 0.0 11.6 (11.5) 24.1 (24.7)

34.4 14.3 45.8 24.2 (28.3) 11.4 (12.1)

14.1 0.0 0.0 0.0 (0.0) 0.0 (0.0)

80 12 — 318 (197) 387 (227)

86.3 91.7 — 84.3 (83.2) 79.1 (79.3)

11.3 0.0 — 13.5 (14.2) 16.8 (17.2)

2.5 8.3 — 2.2 (2.5) 4.1 (3.5)

0.0 0.0 — 0.0 (0.0) 0.0 (0.0)

a

Number of isolates examined. Frequency of chlorate-resistant sectors per colony. Mean, minimum and maximum values. c Percent of chlorate-resistant sectors growing as thin expansive colonies on minimal medium. d Total number of mutants tested. e Mean frequency of mutant phenotypes determined according to growth on basal medium amended with different nitrogen and sulphur sources. f Data within brackets refer to the same 11 and nine isolates tested on the previous media. b

None showed wild-type morphology on taurine, and growth of all sul mutants was inhibited on a medium used to test for resistance to selenate. However, four sul mutants gave rise to 1–4 resistant sectors from six mycelial disks sown, for each mutant, on selenate medium (Table 4). The nit mutants recovered from F. proliferatum were mostly nit1. Very few Nit3 / 8 were observed, and sul mutants were not recovered. Chlorate concentration did not affect the relative frequency of nit mutant phenotypes on PDCL, while MMCL yielded NitM mutants only when supplemented with 4% chlorate (Table 3). Several mutants from both fungi assigned to class Nit3 / 8 were tested for nitrate reductase activity to distinguish between pathway-specific regulatory mutants and nitrite reductase mutants. When tested for nitrite excretion some mutants showed a bright purple color compared to the light purple reaction of the wild-type colony. Such a phenotype corresponded to that described for Neurospora crassa

nitrite reductase mutants (nit6 ) [10,14,18]. This group of mutants is here indicated with the phenotype designation Nit8, as in F. moniliforme nit6 is the designation of one of the five loci coding for a molybdenum-containing cofactor [5]. All strains of F. moniliforme gave rise to nit3 / Nit8 sectors. Out of 96 mutants tested the majority were nit3, while only 6.25% were found to be Nit8. Only nine strains of F. proliferatum produced nit3 / Nit8 sectors, and out of 19 mutants tested 78.9% could be considered Nit8 (Table 5). Intensity of the purple reaction of wild-type colonies was found to be variable in F. proliferatum, so that the purple reaction of Nit8 mutants frequently did not substantially differ from that of the wild types, while nit3 mutants showed none or a very light purple reaction.

3.3. Complementation tests Physiological complementation was positively verified, pairing different nit mutants from the same

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180

Table 4 Growth of sulphate non-utilizing mutants of F. moniliforme on basal medium supplemented with different nitrogen and sulphur sources and mutant sodium selenate sensitivity Media

Mutants

PDA NaNO 3 NaNO 2 Ammonium tartrate Hypoxantine Uric acid Urea Putrescine Asparagine Glutamine Proline Thiosulphate a Cysteine Methionine Taurine Selenate b

Ms4 / 1

Ms5 / 14

Ms5 / 15

Ms6 / 1

Ms10 / 14

Ms11 / 1

Ms14 / 34

R175 / 25

R175 / 31

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 nt

1 2 2 2 2 2 2 2 2 2 2 2 1 1 2 nt

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o

1 2 2 2 2 2 2 2 2 2 2 2 1 1 2 o

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o (0.7)c

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o (0.2)c

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o (0.5)c

1 2 2 2 2 2 2 2 2 2 2 1 1 1 2 o (0.5)c

MM without MgSO 4 replaced by 0.5 g l 2 1 thiosulphate. MM without MgSO 4 amended with taurine 0.1 g l 2 1 and sodium selenate 1 g l 2 1 according to Correll and Leslie [15]. c Mean frequency of resistant sectors per colony is shown within brackets. (o), no growth; (2), starved growth pattern; (1), normal growth; (nt), not tested.

a

b

Table 5 Frequency of nit3 and Nit8 mutants according to a nitrite excretion test [6,18] carried out on most of the Nit3 / 8 mutants from Table 3 F. moniliforme

F. proliferatum

Isolate

Nit3 / 8

nit3

Nit8

Isolate

L1 / 83 C142 / 86 C266 / 86 Ms2 Ms4 Ms5 Ms6 Ms7 Ms8 Ms9 Ms10 Ms11 Ms12 Ms13 Ms14 Ms15 R175 / 87 R176 / 87

3 1 0 6 6 2 15 5 7 3 5 3 3 11 5 10 7 4

1 1 — 6 6 2 15 4 6 2 5 3 3 11 5 10 7 3

2 0 — 0 0 0 0 1 1 1 0 0 0 0 0 0 0 1

A4 A6 A8 C109 / 86 C123 / 86 C219 / 86 C223 / 86 C262 / 86 C212 C237 M1H M1E M3E Ms3 Ms66 Ga63 / 83

Total

96

90

6

nit3

Nit8

1 4 2 5 1 0 2 0 0 0 1 1 2 0 0 0

1 1 0 0 0 — 2 — — — 0 0 0 — — —

0 3 2 5 1 — 0 — — — 1 1 2 — — —

19

4

15

Nit3 / 8

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Table 6 Complementation reactions between sulphate non-utilizing mutants of F. moniliforme and vegetative compatibility of parent isolates

Ms5 / 15 Ms11 / 1 Ms6 / 1 Ms10 / 14 Ms14 / 34 R175 / 25 R175 / 31

Ms5 / 15

Ms11 / 1

Ms6 / 1

Ms10 / 14

Ms14 / 34

R175 / 25

R175 / 31

2c 2i 2c 2c 2c 2i 2i

2i 2c 2i 2i 2i 2i 2i

2c 2i 2c 1c 1c 2i 2i

2c 2i 1c 2c 1c 2i 2i

2c 2i 1c 1c 2c 2i 2i

2i 2i 2i 2i 2i 2c 1c

2i 2i 2i 2i 2i 1c 2c

Complementation reaction: (1), development of dense heterokaryotic mycelium; (2), no reaction observed. Vegetative compatibility: (c), compatible; (i), incompatible [21].

isolate (nit1, Nit3 / 8, NitM) of F. moniliforme and of F. proliferatum. Only the isolate Ga63 gave rise to nit mutants that did not complement. This isolate was considered ‘heterokaryon self-incompatible’ [20]. Some sulphate non-utilizing mutants recovered from F. moniliforme were paired on MM to test for the presence of different mutations, as was also suggested by their different utilization of sulphur sources. Rapid complementation reactions resulting in dense aerial mycelium were observed between some of them. Complementation was observed among Ms6 / 1, Ms10 / 14 and Ms14 / 34 and between R175 / 25 and R175 / 31 (Table 6). Vegetative compatibility between tested isolates is known [21] and is shown in Table 6. Ms5 / 15 behaved differently from its own parent and seemed unable to complement with any of the other mutants from compatible isolates. Data from the remaining mutants suggested they could result from modification of at least three loci associated with sulphate metabolism. Complementation was also verified between sul mutants and all classes of nit mutants. When a sul mutant was paired with one nit1, one NitM or one Nit3 / 8 mutant, recovered from the same isolate, complementation consistently occurred and rapid development of dense heterokaryotic mycelium was observed.

4. Discussion The spontaneous appearance of sectors is common among members of the genus Fusarium [22]. Ge-

netic instability in F. moniliforme has been regarded as associated with transposable elements. It has been proposed that environmental stress, such as growth on a chlorate medium, could trigger the movement of such elements causing a high frequency of spontaneous mutations [5,22]. Daboussi et al. [23] considered transposable element activity a source of genetic variability in F. oxysporum, discovering in this species a family of transposable elements referred to as Fot1, whose first element identified was found as an insertion in the gene encoding nitrate reductase. In this study nitrate non-utilizing (nit) mutants were recovered as spontaneous chlorate resistant sectors from F. moniliforme and F. proliferatum. As with other filamentous fungi, instability on chlorate medium appears to be a general character of strains of F. moniliforme and F. proliferatum. In addition, both the frequency of isolation of chlorate-resistant mutants and the relative percentage of nit mutants was affected by species, isolates and the medium used [2,5,18,24,25]. Nitrogen sources contained in the different media probably influenced the type of nit mutants generated, although the relative frequency of nit mutant phenotypes could also be attributed to differences between loci in susceptibility to mutation [5–7]. Nitrate non-utilizing mutants recovered from F. moniliforme and F. proliferatum were assigned to four phenotypic classes (nit1, nit3, NitM, Nit8), but the relative frequency of these classes was different between the two species. nit1 mutants predominated in F. moniliforme, followed by Nit3 / 8, confirming the observation of Klittich and Leslie [5]. In F. proliferatum, nit1 also prevailed but were followed

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by NitM. Some chlorate-resistant sectors of F. moniliforme were also found to be sulphate nonutilizing mutants (sul). Three phenotypic classes found in our study (nit1, nit3, NitM) corresponded to those assigned in previous studies to mutants of F. moniliforme and F. proliferatum [2,5]. The other two classes (Nit8, sul) were not previously recovered in studies where nit mutants of Fusarium spp. (and of other fungi) were obtained as chlorate-resistant sectors. The presence of sulphate non-utilizing mutants in F. moniliforme and of nitrite reductase mutants (Nit8) in both species is worthy of attention. It is generally believed that mutants in the nitrite reductase structural locus are not commonly recovered when chlorate resistance is used as a screen, since such mutants are usually sensitive to chlorate [6,7,10,24]. Elmer [2] obtained, among others, nit3 mutants from strains of F. proliferatum, but did not test nitrite excretion. Correll et al. [18] recovered 1300 nit mutants from seven strains of F. oxysporum cultured on media amended with 1.5% potassium chlorate. All mutants were divided into three phenotypic classes but none were nitrite reductase mutants. Klittich and Leslie [5] examined the ability of 12 strains of F. moniliforme to sector spontaneously on toxic chlorate medium, finding no mutation affecting nitrite reductase among over 1000 nit mutants. In the latter two studies, mutants were screened for chlorate resistance by growing them, after sector isolation, on media containing potassium chlorate. In our study, on the other hand, we performed no further chlorate resistance tests on our mutants after isolation of sectors from chlorate-containing media. We therefore maintained and characterized all sectors isolated. Brooker et al. [24], working with Colletotrichum, selected their mutants without further testing for chlorate resistance after sector isolation, finding both nit2 and mutants ‘unable to grow well on any of the screening media, including CM’. These results are unusual in Fusarium. Mutants affected in a major nitrogen regulatory locus were also recovered from two strains of F. solani by Hawthorne and ReesGeorge [26]. Similarly to the previous authors, they did not carry out further tests for chlorate resistance after sector isolation. Screening of colonies isolated from fast growing sectors on chlorate medium for

true chlorate resistance could lead to the elimination of those mutations not linked with strong chlorate resistance, such as mutants lacking nitrite reductase [6,14]. However, both Nit8 mutants and sul mutants appeared with a low frequency. Of the total sectors tested, Nit8 mutants were found approximately at a rate of 1.1% in F. moniliforme and 2.5% in F. proliferatum, sul mutants were found only in F. moniliforme and at a rate of 1.2%. Sulphate non-utilizing mutants have been generated on media containing sodium selenate in few fungi other than Fusarium [8,9]. sul mutants were obtained in F. moniliforme and F. oxysporum as fast-growing selenate-resistant sectors, growing as thin expansive colonies on MM but showing wildtype morphology on MM amended with taurine [15,16]. Sulphate non-utilizing mutants described in this paper and isolated as chlorate-resistant sectors differed from those known in F. moniliforme and F. oxysporum. They were unable to utilize taurine, and growth was inhibited by sodium selenate. However, as observed for Aspergillus nidulans [8], the majority of such mutants responded to thiosulphate supplementation, but two mutants required cysteine or methionine to grow with a wild-type morphology. Complementation was also observed when some of the sul mutants were co-cultured on MM. The pattern of positive reaction obtained, even combined with the existence of vegetative incompatibility between isolates [27], suggests that our mutants could result from mutation of at least three loci associated with sulphate metabolism. In A. nidulans, sulphate non-utilizing mutants may result from mutation in one of four loci [28,29], associated with sulphate uptake and reduction of sulphate to sulphite. The exact mechanism of chlorate toxicity is unknown. The simplest hypothesis, that chlorate was rendered toxic by nitrate reductase conversion to chlorite, is almost certainly correct but insufficient to explain all the data available [10]. Data collected here show that in F. moniliforme and F. proliferatum, classes of mutants other than those already reported can be obtained on chloratecontaining media. However, they appear at low frequency and detection is affected by the selection method used.

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On the other hand, culturing F. moniliforme on chlorate medium can result in genetic instability that may be associated with a transposable element [5]. Recently, Anaya and Roncero [30] observed rearrangement of the retroelement skippy in F. oxysporum f.sp. lycopersici grown under stress conditions in the presence of potassium chlorate. Consequently, the unexpected mutants found in F. moniliforme in our study could be associated with transposable elements. As transposon movement can cause a high frequency of mutations, it should not be surprising to find other mutants on chlorate-containing media beyond those strictly linked to high chlorate resistance. In F. proliferatum the range of mutants detected was lower compared with that of F. moniliforme and no data are yet available on transposable elements in this species, however their presence should not be excluded.

Acknowledgements Research work has been supported by C.N.R., Research Group ‘Patologia delle Piante Ortensi’, publication no. 322. We wish to thank Prof. Piero Gambogi for helpful discussions and Maurizio Forti for his skilled technical assistance.

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