Mycol. Res. 97 (I): 59-67 (1993) (Printed in Great Britain)

A new species of Sphaerulina and its Phloeospora anamorph,with potential for biological control of Mimosa pigra

HARRY C. EVANS1, GLORIA CARRION2 AND GASTON GUZMAN2

1 International Institute of Biological Control (lIBC), Silwood Park, Ascot, Berks. SL5 7TA UK2 Proyeeto Hongos, Instituto de Ecologia (lE), Apartado Postal 63, Xalapa, Veracruz, Mexico

Sphaerulina mimosae-pigrae and its coelomycete anamorph, Phloeospora mimosae-pigrae, are described as new. The fungus occurs ongreen leaf pinnae, rachides, branches and stems on Mimosa pigra in tropical America, causing extensive necrosis and die-back.Collections from Mexico, Trinidad, Venezuela, Colombia and Brazil are recorded and compared. The taxonomic status and biologyof the pathogen are discussed, and its potential as a biological control agent of Mimosa pigra in Australia is evaluated.

59

Mimosa pigra L. (Mimosaceae), or giant sensitive plant, is atropical, leguminous, spiny shrub with a native geographicalrange extending from southern Texas to southern Brazil. Thecentre of origin is purported to be Central America, possiblyMexico (Lonsdale & Braithwaite, 1988; Lonsdale, Harley &

Gillett, 1988; Lonsdale, Miller & Forno, 1990), but morerecent field surveys, as well as taxonomic investigations(Barneby, 1989), have shown that this may be a rathersimplistic interpretation of its evolutionary history. In Mexico,M. pigra is a relatively uncommon plant, being restricted tolowland swampy areas along both the Gulf and Pacific coasts,where two varieties have been recognized: M. pigra var. pigraL. and vaL berlandieri (A. Gray ex TorL) B. L. Turner. However,the nomenclatural and taxonomic status of the M. pigracomplex is currently uncertain (Barneby, 1989; Verdcourt,1989).

In endemic, neotropical habitats, M. pigra seldom reachesmore than 2 m in height (Janzen, 1983), but commonly growsup to 5-6 m in exotic situations, such as northern Australiaand Thailand, forming dense impenetrable thorn thickets(Lonsdale & Braithwaite, 1988; Lonsdale ef al., 1990). Thisincreased fitness of the plant in alien habitats is, in the main,considered to be due to the absence of natural enemies.Biological control, through the deliberate introduction of co­evolved natural enemies, is seen to offer the best means oflong-term or sustainable management of this weed, particularlyin the Northern Territory of Australia where the continual useof chemical herbicides over vast areas, such as national parks,is both uneconomic and environmentally undesirable(Braithwaite, Lonsdale & Estkergs, 1989; Lonsdale ef al., 1990).

An entomological research programme has been operating inMexico since the early 1980s, searching for and screeningpotential insect control agents. Several insect species, includingseed-eating bruchids, have already been introduced into

Australia (Lonsdale & Braithwaite, 1988), whilst others,including flower-feeders, are still being evaluated (Lonsdaleef al., 1990). In order to increase the biocontrol options againstthis difficult target, searches for fungal pathogens wereinitiated in 1988. Several candidate agents are currently underinvestigation. This paper reports on one of these, consideredto represent an undescribed taxon.

MATERIALS AND METHODS

Field surveys

Mimosa pigra stands were surveyed along both the Gulf andPacific coasts of Mexico during the dry and wet seasons of1988 and 1989. Later, ad hoc collections were made inColombia (1991) and Brazil (1991), and by collaborators inTrinidad (1990, 1991) and Venezuela (1990, 1991). Diseasedmaterial was collected from foliage, inflorescences and stems,air-dried for 2-3 d in a plant press, and stored in waxedpackets prior to examination. In addition, searches were madeon related Leguminosae in general, in order to provisionallyassess the natural host range.

Laboratory studies

Material with sporulating conidiomata was immersed for5 min in sterile distilled water and the resultant sporesuspension was spread over tap-water agar (TWA) in Petridishes with a glass rod. Individual perithecia (ascomata) werepicked-off with the aid of a fine hypodermic needle using astereoscopic microscope and squashed directly on to TWAplates. Germinating spores were removed aseptically after24-48 h at 20-25 °C and streaked on to TWA containingstreptomycin sulphate and penicillin. Established colonies

Sphaerulina sp. nov. on Mimosa pigra

were transferred to potato carrot agar (PCA) and potatodextrose agar (PDA) and incubated at 25°.

RESUL TS AND DISCUSSION

Taxonomy

Sphaerulina mimosae-pigrae Evans & Carrion, sp. nov.(Figs 1-10)

Status anamorphicus: Phloeospora mimosae-pigrae Evans &Carrion, sp. nov. (Figs 11-20)

Maculae in JoWs amphigenae, discretae, ovoideae, badiae vel obscurebrunneae; maculae in ramis obscure brunneae vel atrae, restrietae adextensus, emortescentes. Ascomafa in ramis (rhachidibus), erumpentia(superficialia in apparencia), conidiomata insidentia, dissita velgregaria, glabra, obscure brunnea vel atra, globosa vel subglobosa,120-200 x 100-180 I-lm, ostiolata, periphysata; paries 14-20 I-lmcrassus usque ad 26 I-lm, mulhstratus ex cellulis pseudoparenchymatis.Asci cylindrico-clavati, 65-105 x 15-20 I-lm, octospori, crassitunicati,bitunicati. Ascosporae 2-3 seriatae, fusiformes, laeves, hyalinae, 2-3septatae, guttulatae, 22-32 X 5-8 I-lm. Conidiomafa acervularia, soli­taria vel gregaria, alba, dehiscentia irregularia, 10D-500 x 100­180 I-lm, tenuitunicata. Cellulae conidiogenae holoblasticae, sympo­diales, hyalinae, indeterminatae, leviter flexuosae, (14-) 18-24(-28) x 3-5 I-lm. Conidia hyalina, cylindrica, 3-11 septata, apicerotundata ad basim obtusa vel truncata, (33-) 44-56(-80) x 3-6 (-8) I-lm, formata in columnis mucosis, albis, usque ad1'5 mm longis. Spermogonia praesens, affinis Asferomellae; spermatiahyalina, bacilliformia, 4-5 x 1 I-lm.

Holotypus: !M! 340371, in foliis, leguminibus, ramis et caulis vivis exMimosae pigrae L., Chontalpa, Tabasco, Mexico, 19 Nov. 1988,H. C. Evans et G. Carrion; isotypus Herb. XAL.

Leaf lesions amphigenous, discrete, circular to ovoid, up to2 mm diam., chocolate to dark brown; lesions on branches andstems, occasionally on pods, spreading and coalescing to formdark brown to black extensive cankers. Ascomata on branches(rachides) only, subepidermal, erumpent but appearing super­ficiaL forming around edges of conidiomata, scattered toaggregated, glabrous, dark brown to black, globose tosubglobose, 120-200 x 100-180 I-lm, ostiolate, periphysate;wall multi-layered, composed of pseudoparenchyma, 14­20 I-lm thick, up to 26 I-lm. Asci cylindrical to clavate,65-105 x 15-20 I-lm, 8-spored, thick-walled, bitunicate, matur­ing sequentially. Ascospores 2-3 seriate, fusiform, smooth,hyaline, guttulate, 2-3 septate, 22-32 x 5-8 I-lm.

Conidiomata acervular, subepidermal, solitary to gregarious,white, dehiscence irregular, 100-500 x 100-180 I-lm, thin­walled. Conidiogenous cells sympodial, hyaline, indeterminate,slightly flexuous, (14-) 18-24 (-28) x 3-5 I-lm, with 1-3unthickened scars. Conidia holoblastic, hyaline, cylindrical, 3­I I septate, with rounded apex and obtuse to truncate base,(33-) 44-56 (-80) x 3-6 (-8) I-lm, guttulate when young;formed in dense, white, mucous-covered columns, up to1'5 mm long. Spermogonia present, Asteromella type; spermatiahyaline, bacilliform, 4-5 x I I-lm.

Specimens examined: Holotype: !M! 340371, on living leaves, pods,branches and stems of Mimosa pigra L., Chontalpa, Tabasco, Mexico,19 Nov. 1988, H. C. Evans & G. Carrion; isotype Herb. XAL.Parafypes: (Iectus H. C. Evans, except where stated). Mexico:!M! 340373, Villahermosa, Tabasco, 27 Feb. 1988; !M! 340372, Las

60

Piedras, Veracruz, 25 Feb. 1988; !M! 340370, Paso del Toro,Veracruz, 23 Nov. 1989; !M! 340375. Cuajinicuilapa, Guerrero. 30Nov. 1%9; in Herb. XAL; Paso del Toro, 27 Sep. 1989, G. Carrion;Paso del Toro, 12 Dec. 1989, F. Ruiz-Belin. Brazil: !M! 352344, RioArara-Rio Negro, Amazonas, 27 July 1991; !M! 352347, Rio Pardo,Bahia, 9 Aug. 1992; !M! 352345. Rio Macapuruna-Rio Solimoes,Amazonas. 30 July 1991. Colombia: 1M! 352350. Rio Tobia.Cundinamarca, 21 July 1991. Venezuela: !M! 352351, Bruzua!,Apure, 19 Nov. 1990, I. W. Forno; !MI 352352, Apurito, Apure, 21Nov. 1990, I. W. Forno; !MI352353, Achagua, Apure, 23 Nov.1990, I. W. Forno; 1M! 352354, Guayaba!, Guarico, 27 Nov. 1990,I. W. Forno; !M! 352355, Manteca!, Apure, 14 Nov. 1991,I. W. Forno; !M! 352356, Guasdalito, Apure. 12 Nov. 1991,I. W. Forno.

Other material examined: (Iectus H. C. Evans, except where stated,anamorph only). Mexico: 1M! 304382, Villahermosa, Tabasco, 18Mar. 1986, R. Kassulke; 1MI 340350, Coatzalcalcos, Tabasco, 26 Feb.1988; !M! 340351, Villahermosa, Tabasco, 27 Feb. 1988;!M! 340349, Paso del Toro, Veracruz, 29 Feb. 1988; !M! 340348, RioTonahi-Las Choapas, Tabasco, 19 Nov. 1988; 1M1 340352, BenitoJuarez-Simon Sarlat, Tabasco, 26 Nov. 1988; !M! 340354, PuenteGuasimil, Veracruz, 6 May 1989; !M! 340353, Tuzales-PiedrasNegras, Veracruz, 7 May 1989; !M! 340356. Puerto Lobos, Alvarado,Veracruz, 23 Nov. 1989; !M! 340355, Puente Rio Verde, Oaxaca, 1Dec. 1989; !M! 352341, Lorna Bonita, Veracruz, 24 Feb. 1991: inHerb. XAL; Puente Tlacotalpan, Veracruz, 28 Oct. 1988, F. Ruiz­Belin; 1sla-Acayucan, Veracruz, 3 Dec. 1989, G. Carrion; Atasta,Campeche, 26 Nov. 1988, F. Ruiz-Belin. Brazil: 1M! 352343, PuntaNegra, Manaus, Amazonas, 29 July 1991; !M! 352342, Macapuruna,Amazonas, 30 July 1991; 1M! 352346, Rio Siqueira, Fortaleza, Ceara,6 Aug. 1991. Colombia: !M! 352359, Monte Lindo, Palestina,Caldas, 14 Feb. 1991. Trinidad: !M! 337430, St Augustine, Jan. 1990,C. Prior; !M! 352348. May 1991, C. Prior.

Culture: ex IMI 340355, ascospore isolate; on PCA and PDA,slow-growing (0·4-{}6 em diam. after 14 d, 1'0-1'2 em after28 d), pink, stromatic, convoluted, yeast-like, consisting ofbudding conidia (Fig. 20), becoming dark pink to orange­brown with age. Spermogonia formed after 6 wk on PCA inblack light; globose, fleshy, hyaline to pale brown. Conidialisolates: 1M1352349, 352350, 352344, 337430, 340370,

340354, 340356, 352353, 352347. Some cultures showmycelial sectoring with age.

IMI 352350 from Colombia has densely packed ascomatacovering the rachides and conidia which are consistentlylonger than the type (range 55-85 ~m, average length68·5 1Jffi). Ascomata frequently unite to form multiloculatestructures (Fig. 4). Conversely, the other Colombian specimen(IMI 352349) has significantly shorter conidia than the othercollections (range 24-48 I-lm, average length 35 I-lm) (Figs15-16). There is a similar discrepancy in spore size amongstthe Brazilian specimens, those from Central Brazil (Bahia)having longer conidia (e.g. IMI 352347; range 47-90 I-lm,average length 65'5 I-lm) than Amazonian material (e.g.IMI 352344; range 35-70 I-lm, average length 51'5 I-lm) (Fig.17). However, until the taxonomy of the M. pigra complex isfinally resolved and reciprocal pathogenicity tests have beencompleted, separation at the varietal (or higher) level is notadvisable.

The genus Sphaerulina Sacc. is considered by Barr (1972) tobe derived from the section Longispora of Mycosphaerellasubgen. Mycosphaerella, being separated on the basis of the

H. C. Evans, Gloria Carrion and G. Guzman 61

Figs 1-5. 5phaerulinamimosae-pigrae(1M! 340371). Fig. 1. T.S. of mature, erurnpent ascoma. Bar = 20 Ilm. Fig. 2. T.S. of immatureascoma with ascus mother cells. Bar = 20 Ilm. Fig. 3. Dark brown to black, globose, ascomata amongst white acervuli and plant hairson rachis. Bar = 250 Ilm. Fig. 4. T.S. to show ascomata uniting to form a multiloculate structure. Bar = 40 Ilm. Fig. 5. Mature asci. Bar= 10 I-lill.

multiseptate, elongate ascospores. Barr (1972) distinguishedtwo groups within Sphaerulina: ascomata maturing in dead,overwintered leaves; fertile ascomata developing in lesions onliving leaves. The species on M. pigra falls within the lattergroup in that the ascomata are associated with living hosttiss'les, although these never form on true leaf tissue (leaflets)but on the woody rachides (both primary and secondary).Sphaerulina spp. are typically foliar pathogens with anamorphsin the genera Septoria (as Cylindrosporium or Phloeospora),Cercospora and Cercosporella (Barr, 1972; Arx & Muller, 1975;

Sivanesan, 1984). Evans (1987) failed to record any Asco­mycetes on M. pigra on the basis of herbarium and literaturesurveys. However, a subsequent literature search revealed thatMycosphaerella mimosicola P. Henn. had been described onMimosa asperata L. from Brazil (Hennings, 1904). M. asperatais a synonym of M. pigra and, indeed, has been proposed asthe earlier valid name (Bameby, 1989). The description of theascospores (I-septate, hyaline, medianly constrided) fits thatof the genus Mycosphaerella, and similar material has beencolleded recently by the authors from both Mexico and

Sphaerulina sp. nov. on Mimosa pigra 62

Fig. 7. Ascospores from a single ascus; typically 3-septate, guttulate (Nomarski, bar = 10 IJm). Figs 8-10. Stages in maturation of asci(Nomarski, bar = 10 IJm).

Brazil. No species of Sphaerulina or Phloeospora has beenrecorded previously on Mimosa, nor apparently on anymember of the Mimosaceae (fide Saccardo's Sylloge Fungorum(1882-1931), Petrak's Lists, 1920-1939; Index of Fungi,1940-1992).

Diagnoses of Sphaerulina describe the ascomata as immersedor erumpent, frequently stromatic (Barr, 1972; Sivanesan,

1984), whilst Arx & Muller (1975) key out the genus on thebasis of its small, discrete ascomata, immersed in the hosttissue. In contrast, ascomata of the Mimosa pathogen arestrongly erumpent, appearing almost superficial (Fig. 25), andstromatic when produced in abundance. Nevertheless, based onascus and ascospore morphology, the fungus can only be ac­commodated within Sphaerulina. Most of the species aSSigned

H. C. Evans, Gloria Carrion and G. Guzman 63

Figs 11-14. Phloeospora mimosae-pigrae (IMI 340371). Figs 11-13. T.S. through rachides showing erumpent, subepidermal acervuli (Figs11-12, bar = 100 pm, Fig. 13, bar = 50 1ill1), Fig. 14. T.S. through acervulus showing sympodial conidiogenous cells. Bar = 10 pm.

to the genus have been examined in Herb. IMI (18 spp.)and S. mimosae-pigrae can be separated readily on ascomatalform and habit. Similar problems surround the correctplacement of the anamorph. Using the Coelomycete keys ofSutton (1980), the anamorph, with its acervular conidiomata,sympodial conidiogenous cells and cylindrical multiseptateconidia can be placed in Phloeosporella Hahn. However, thereis considerable confusion between Phloeosporella and thegenera Cylindrosporium aud., Septogloeum and Phloeospora.Sutton (1980) rejected the inclusion of the anamorph ofMycosphaerella mori (FuckeI) Wolf in Phloeosporella, as proposedby Boerema & Verhoeven (1973), and referred it to Phloeospora,as P. maculans (Bereng.) Allesch., on the basis of its annellidicconidiogenous cells. Phloeospora is a large genus, with over160 species, containing several important plant pathogens,including the aforementioned leaf spot disease of mulberry(Smith & Stewart, 1947). In the generic diagnosis (Sutton,1980), the conidiogenous cells are described as anneIIidic butoccasionally also sympodiai. It is concluded here thatPhloeosporella should be reserved for anamorphs of Heliotales,since Blumeriella jaapii (Rehm) Arx has been shown to be theteleomorph of Phloeosporella padi (Lib.) Arx (Arx, 1961). Morerecently, Williamson & Bernard (1988) have endorsed thisholomorph association by describing a new Blumeriella species

with a Phloeosporella anamorph, which they transferred fromCylindrosporium. Phloeospora, therefore, represents an ana­morphic genus of fungi with Mycosphaerella-like teleomorphs(Dothideales).

Biology

Field surveys indicate that S. mimosae-pigrae is specific toMimosa pigra sensu lata, particularly since closely relatedMimosaceae, such as M. invisa Mart. and M. pudica L.,growing in close association with M. pigra never showedevidence of infection by this fungus. Host range tests, usingthe centrifugal phylogenetic method devised by Wapshere(1974, 1989), are currently under way to confirm this fieldspecificity. More than 90 test species are being screened,including a large representative selection of Australian Acaciaspp. The taxonomic position of the pathogen has, to a certainextent. determined the composition of the test plant list, asplants of agricultural importance in Australia which are knownhosts of Phloeospora spp. have also been included.

It is also evident from the results of the survey that S.mimosae-pigrae is a Widespread pathogen of M. pigra, probablyoccurring throughout the natural range of the plant in theNeotropics. It is surprising, therefore, 'that the fungus has not

5phaerulina sp. nov. on Mimosa pigra 64

Figs 15-20. Phloeospora mimosae-pigrae (all Nomarski, bar = 10 J-lm). Figs 15-16. Conidiogenesis in IMI 352349, ex Colombia, showingshort conidial form. Fig. 17. Conidia from IMI 352344, ex Amazonia (Brazil). Figs 18-19. Conidiogenesis and conidial form inholotype (IMI 340371) ex Mexico. Fig, 20. Budding conidia from culture, note mucoid covering (arrow).

been described previously, particularly since both the advancedsymptoms and the plant are conspicuous. Symptomatology isunusual, however, and has been confused in earlier ento­mological surveys with mealybug infestations. Plant patho-

gens pertaining to the genera 5phaerulina and Phloeospora areassociated with foliar diseases, typically causing leaf spotting(Barr, 1972; Sutton, 1980; Sivanesan, 1984; Hanlin, 1990). 5.mimosae-pigrae can produce discrete leaf spots on its host. but

H. C. Evans, Gloria Carrion and G. Guzman 65

Figs 21-25. Sphaerulina mimosae-pigrae. Fig. 21. Die-back of rachis, Veracruz, Mexico. Note the dense white sporulation. Fig. 22. Stemcanker and Fig. 23. Rachis die-back, showing dense. white, mucilaginous, conidial columns. Bar = 250 1llJl. Fig. 24. T.S. through youngacervulus. Bar = SO 1llJl. Fig. 25. T.S. through old acervulus showing development of ascoma at edge. Bar = SO 1llJl·

MYC 97

Sphaerulina sp. nov. on Mimosa pigra 66

Figs 26-30. Sphaerulina mimosa-pigrae. Figs 26-27. Heavily infected stand of Mimosa pigra, Rio Arara, Amazonas, Brazil. The plantstypically grow at the edge of the flooded rivers (varzea) and are fully exposed in the dry season. Note spindly habit and severedefoliation associated with branch and stem cankering. Figs 28-29. Acervuli on green rachides producing columns of hyaline conidia,extending along the rachis (arrow). Bar = 0'5 mm. Fig. 30. Ascomata on necrotic rachis; the conidia have been spread over the surfacefollowing rain. Bar = 250 1IJIl.

H. C. Evans, Gloria Carrion and G. Guzman

this symptom is invariably secondary compared with theextensive cankering of the leaf rachides, branches and stems,leading to progressive dieback and plant decline (Figs 22, 26,27). Nevertheless, at one locality (lMI340352), spotting ofthe leaflets was common and damaging with an absence ofsymptoms on other tissues. It is uncertain if this is due topathogenic or phenological differences. Greenhouse inocula­tions have shown that the incubation period, from hostpenetration to symptom expression, is prolonged (ca 4-5 wk).Sporulation is initially on green tissues and frequentlysynchronous along the primary and secondary rachides (Figs21, 28). This suggests that the fungus colonizes the tissuessystemically and that, during this prolonged asymptomlessphase, it has a biotrophic mode of existence. Nutritionally,therefore, S. rnirnosae-pigrae can be classified as a hemibiotroph(Lewis, 1973; Luttrell, 1974). Roivainen (1977) reported aspecies of Phloeospora, P. (as' Phloeospora') aenigrnafica Rov.,which apparently systemically infected its host, causing shootabnormalities, and considered this to be highly unusual for aCoelomycete (' scolecosporous ') fungus.

A further distinguishing feature of S. rnirnosae-pigrae is itssporulation habit. The hyaline conidia exude in long, dense,mucilaginous columns from acervuli on rachides, branches andstems which are freely exposed to environmental conditions(Figs 23, 29). These are extreme in many of the habitatssurveyed, particularly along the Pacific coast of Mexico wherethere is a marked dry season. Conidial material collectedduring the dry season from such localities (IMI 340355), andwhich must have been exposed over a period of severalmonths to high levels of solar radiation, germinated readily onTWA. In general, stored herbarium material retained anacceptably high level of germination (60-80%) for up to 6months. The conidial mucilage must contain, therefore, aneffective sun-blocking component, as well as a dispersant; thespores exhibit considerable Brownian movement when wetted.

In several collections (IMI352350, 352351, 352347,340370, 352352, 352353, 352354), the teleomorph isdominant the living rachides being covered by masses of darkbrown ascomata (Fig. 30). The regular occurrence of theteleomorph in diverse localities suggests that it plays animportant role in the life cycle and, in terms of its potential asa classical biological control agent, this would be a highlydesirable character. Traditionally, the fungal groups exploitedas classical agents have been obligate pathogens, such as rustsand smuts, with highly efficient spore-dispersal mechanisms(Evans & Ellison, 1990). Coelomycetes, especially those withslime spores, have not been considered suitable for theclassical approach due to poor dispersal ability, although themycoherbicide approach has been or is being developed forCoe1omycete genera, such as Collefofrichurn and Phornopsis(Charudattan, 1991). Thus the presence of both an anamorph,with abundant slime-spored conidia, for rapid short-distancemovement by rainsplash or run-off, and a teleomorph, withaerially dispersed dry spores, would ensure efficient spread ofthe pathogen within and between weed populations.

This project was funded by the Australian Government

(Accepted 28 August 1992)

67

(ACIAR-CSIRO). Thanks are due to Georgina Godwin andPaul Cripps for photographic assistance.

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