Phylogenetic analysis
Twelve Nigrospora isolates obtained as endophytes from Manihot esculenta were identified by phylogenetic analysis using sequences of ITS, TEF1- α and TUB2 genic regions. Phylogenetic analyses were first performed for each genic region individually (data not shown), and then concatenated in the same matrix used to construct a consensus phylogenetic tree based on Bayesian inference. The concatenated alignment contained 1376 characters including gaps (491 ITS; 523 TEF1- α; 362 TUB2), 62 taxa. Apiospora malaysiana CBS 102053, and A. pseudoparenchymatica LC7234 were used as an outgroup.
According to the concatenated gene tree, seven isolates grouped with species already known - N. hainanensis (4), N. lacticolonia (1), N. pyriformis (1) and N. vesicularifera (1). This is the first time these species have been identified from M. esculenta. Five isolates did not cluster with any knows species of Nigrospora, and are new to the genus. They formed three new clades well supported with Bayesian posterior probability (BPP) value of 1 on the tree (Fig. 1). The novel species are described below as N. endophytica, N. manihoticola and N. pernambucoensis.
Taxonomy
Nigrospora endophytica A.C.Q. Brito & A.R. Machado, sp. nov. Figure 2
MycoBank number: MB845749
Type: BRAZIL, Pernambuco state, Condado municipality, in cassava field, 7° 35′18.959″ S 35° 4′ 59.022″ W, alt. 118 m, isolated as an endophyte from stem of Manihot esculenta Cranz, Jan 2020, A.C.Q. Brito, Holotype (URM94837), Ex-type culture (URM8462 = ARM973), Genbank accession numbers: OM265233 (ITS), OP572416 (TEF1- α), OP572420 (TUB2).
Etymology
Named in reference to the endophytic life style of this fungus.
Description: Sexual morph not observed. Asexual morph: Hyphae septate, smooth, branched, sometimes coiled, hyaline or pale brown to dark brown, 2.5–7.5 µm wide (av. 4.6 µm). Conidiophores rare, not different from vegetative hyphae, predominantly reduced to conidiogenous cells. Conidiogenous cells monoblastic, determinate, solitary, smooth, pale brown or dark brown, globose, 6.2–10 µm diameter (av. 7.4 µm). Conidia acrogenous, solitary, simple, smooth, shiny, aseptate, pale brown to dark brown, globose or subglobose, 10–17.5 µm diam (av. 13.2 µm). Culture characteristics on PDA, rapid growth reaching 9 cm diameter in 7 days; colonies wooly, flat, edge entire; at first pale olivaceous grey in the center of the colonies with margin grayish white becoming completely olivaceous grey in 12 days (25°C, in the dark); reverse iron grey to grey olivaceous from center towards edge of the culture with smoke gray margin becoming entirely black in 12 days.
Habitat and known distribution
At the moment, Nigrospora endophytica is only found as an endophyte from internal tissues from the stem of Manihot esculenta Cranz. Its distribution is restricted to cassava fields in Condado and João Alfredo municipalities, Pernambuco state, northeast of Brazil.
Other material examined: BRAZIL, Pernambuco state, João Alfredo municipality, in cassava field, 7°49′40.6″S 35°33′13.5″W, approximately 87.2 Km of distance from ex-type location, also isolated as an endophyte from stem of Manihot esculenta Cranz, Oct 2019, S. S. Nascimento, (ARM687).
Notes: Nigrospora endophytica clustered in a well-supported clade, sister to N. pernambucoensis sp. nov (Fig. 1). In the phylogenetic analysis of genes individually, N. endophytica and N. pernambucoensis are closely related according to ITS genic region. Morphologically, N. endophytica showed very little spore production, taking much longer for conidia formation compared to N. pernambucoensis under the same culture conditions (WA culture medium, 25° C, in the dark). Conidiogenous cells and conidia occurring isolated and dispersed along the hypha in N. endophytica (Fig. 2), unlike in N. pernambucoensis where the hyphae develop clusters with formation of conidiogenous cells and conidia close to each other in a coiled manner (Fig. 4). Only globose conidiogenic cells were observed in N. endophytica, while N. pernambucoensis presented more than one shape of conidiogenic cells. Conidia size is smaller in N. endophytica (10–17.5 µm diam) than N. pernambucoensis (12.5–20 µm diam).
Nigrospora hainanensis M. Wang & L. Cai, Persoonia 39: 136 (2017).
Description of the species
Wang et al (2017).
Type
CHINA, Hainan Province, on leaf of Musa paradisiaca, 21 Sept 2015, F. J. Liu, Holotype (HMAS 247064), Ex-type culture (CGMCC3.18129 = LC7030).
Material examined
BRAZIL, Pernambuco state, Condado municipality, in cassava field, 7° 35′ 18.959″ S 35° 4′ 59.022″W, alt. 118 m, isolated as an endophyte from stem of Manihot esculenta Cranz, Jan 2020, A.C.Q. Brito, (ARM967, ARM968, ARM972, ARM976).
Notes
Nigrospora hainanensis has a known distribution mainly in Chinese provinces in different hosts, Musa paradisiaca, Saccharum officinarum, and Oxalis corymbosa (Wang et al. 2017; Raza et al. 2019; Zheng et al. 2021). Outside China, N. hainanensis was previously identified in Brazil, in Napolea cochillifera, as pathogen associated with cladode brown spot (CBS) (Conforto et al. 2019). In the present study, we identified five N. hainanensis strains as endophytes in a new host Manihot esculenta Cranz. In terms of geographical location, N. hainanensis isolates obtained by Conforto et al (2019) and in our study were identified in the same Brazilian state (Pernambuco). It is possible to observe that the species has no host specificity, and can be found in the environment both as a pathogen, and as an endophyte. So far, it appears that the known distribution of N. hainanensis is limited to China and Brazil.
Nigrospora lacticolonia M. Wang & L. Cai, Persoonia 39: 131 (2017).
Description of the species
Wang et al (2017).
Type
CHINA, Jiangxi Province, on Camellia sinensis, 24 Apr 2013, F. Liu, Holotype (HMAS 247070), Ex-type culture (CGMCC 3.18123 = LC3324).
Material examined
BRAZIL, Pernambuco state, Jucati municipality, in cassava field, 8° 47′ 15.1″ S 36° 25′ 59.407″ W, alt. 706, isolated as an endophyte from stem of Manihot esculenta Cranz, Dec 2019, A. R. Machado, (ARM921).
Notes
The first occurrence of Nigrospora lacticolonia, in the Neotropical region, was on healthy leaves of Guarea macrophylla, collected in a Cocoa agroecosystem in Brazil (Santos et al. 2021). In our study, one strain of N. lacticolonia was identified on healthy stem of Manihot esculenta Cranz. Thus, this is the first record of N. lacticolonia in this host in the world, and this may also be a second report of this species in the Neotropical region. Previous records of N. lacticolonia have been reported in China on Camellia sinensis, Musa paradisiaca, and Saccharum officinarum (Wang et al. 2017; Raza et al. 2019), in Malaysia on Hylocereus polyrhizus (Kee et al. 2019), and in Oman on Phoenix dactylifera L. (Al-Nadabi et al. 2020). The lifestyle of N. lacticolonia can be endophytic (Santos et al. 2021) or pathogenic on plants (Raza et al. 2019; Kee et al. 2019; Al-Nadabi et al. 2020).
Nigrospora manihoticola A.C.Q. Brito & A.R. Machado, sp. nov. Figure 3
MycoBank number: MB845748
Type: BRAZIL, Pernambuco state, Chã Grande municipality, in cassava field, 8° 13′46.37″ S 35° 27′ 14.874″W, alt. 505 m, isolated as an endophyte from stem of Manihot esculenta Cranz, May 2019, A.C.Q. Brito, Holotype (URM94836), Ex-type culture (URM8461 = ARM645), GenBank accession numbers: OM265224 (ITS), OM914791 (TEF1- α), OM869479 (TUB2).
Etymology
Named in reference to the host from which it was isolated Manihot esculenta Cranz.
Description: Sexual morph not observed. Asexual morph: Hyphae septate, smooth, branched, sometimes coiled, hyaline or pale brown to dark brown, 2.5–7.5 µm wide (av. 2.95 µm). Conidiophores rare, short, not different from vegetative hyphae, predominantly reduced to conidiogenous cells. Conidiogenous cells monoblastic, determinate, solitary, smooth, pale brown or dark brown, globose to subglobose to ovoid, 3.7–15 µm length × 3.7– 12.5 µm width (av. 6.7 × 6.8 µm). Conidia simple, smooth, solitary, acrogenous, aseptate, shiny, dark brown to black, globose to subglobose to ellipsoidal, 10–17.5 µm diam (av.12.41 µm). Culture characteristics on PDA, the colony exhibit rapid growth reaching 9 cm diameter in 7 days; edge entire with floccose appearance, initially white becoming grayish in 12 days (25°C, in the dark); reverse white with greenish black patches that spreads across the culture, turning the reverse fully black with age.
Habitat and known distribution
At the moment, Nigrospora manihoticola is only found as an endophytic from internal tissue from stem of Manihot esculenta Cranz. Its distribution is restricted to cassava field in Chã Grande municipality, Pernambuco state, Northeast of Brazil.
Notes
Nigrospora manihoticola clustered in a well-supported clade close to N. hainanensis in accordance with the genes individually and the concatenated gene tree (Fig. 1). Morphological differences between these two species can be seen in the hyphae, conidiogenous cells, and conidia. Nigrospora manihoticola exhibits slightly wider hyphae, and their coloration is predominantly pale brown and dark brown, and in N. hainanensis they are hyaline to pale brown. Coiled hyphae are present in N. manihoticola with the production of conidiogenous cells and conidia, but such feature is absent in N. hainanensis. The conidiogenous cells are hyaline with a globose or ampulliform shape in N. hainanensis (6.5–12.5 × 4.5–9.5 µm), while on N. manihoticola was observed pale brown or dark brown with globose to subglobose to ovoid shape (3.7–15 × 3.7– 12.5 µm). In addition to black conidia present in both species, N. manihoticola also displays dark brown conidia. Another morphological difference is the presence of setae that occurs on N. hainanensis, and was not observed on N. manihoticola (Wang et al. 2017). N. hainanensis was isolated from leaves of Musa paradisiaca in China (Wang et al. 2017), and has been associated with plant diseases in more than one host (Conforto et al. 2019; Raza et al. 2019; Zheng et al. 2021), while N. manihoticola was identified as endophytic lifestyle in internal tissues of the stem of Manihot esculenta Cranz in Brazil.
Nigrospora pernambucoensis A.C.Q. Brito & A.R. Machado, sp. nov. Figure 4
MycoBank number: MB845747
Type: BRAZIL, Pernambuco state, Condado municipality, in cassava field, 7° 35′ 18.959″ S 35° 4′ 59.022″ W, alt. 118 m, isolated as an endophyte from stem of Manihot esculenta Cranz, Jan 2020, A.C.Q. Brito, Holotype (URM94838), Ex-type culture (URM8463 = ARM974), GenBank accession numbers: OM265234 (ITS), OM914793 (TEF1- α), OM869481 (TUB2).
Etymology
Named in reference to the Brazilian state, Pernambuco, where the type was collected.
Description: Sexual morph not observed. Asexual morph: Hyphae septate, smooth, highly branched, sometimes coiled, hyaline to pale brown to dark brown, 2.5–6.2 µm wide (av. 3.7 µm). Conidiophores rare, short, not different from vegetative hyphae, predominantly reduced to conidiogenous cells. Conidiogenous cells monoblastic, determinate, discrete, solitary, smooth, pale brown to dark brown, globose to obpyriform 5– 22.5 µm length × 5–12.5 µm width (av. 8.5 × 6.8 µm). Hyaline vesicles around the septum delimiting the conidia and their conidiogenous cells. Conidia acrogenous, solitary, simple, smooth, shiny, aseptate, globose or ellipsoidal, pale or dark brown to black, 12.5–20 µm diam (av.14.9 µm). Culture characteristics on PDA, colonies wooly, edge entire, fast growing reaching 9 cm diameter in 7 days, initially pale olivaceous grey become olivaceous grey in 12 days (25°C, in the dark); reverse iron grey to grey olivaceous from center towards edge of the culture with olivaceous buff margin becoming entirely black in 12 days.
Habitat and known distribution
At the moment, Nigrospora pernambucoensis is only found as an endophytic from internal tissues from stem of Manihot esculenta Cranz. Its distribution is restricted to cassava fields in Condado and João Alfredo municipalities, in Pernambuco state, Northeast of Brazil.
Other material examined
BRAZIL, Pernambuco state, João Alfredo municipality, in cassava field 7°49′40.6″S 35°33′13.5″W, approximately 87.2 Km of distance from ex-type location, also isolated as an endophyte from stem of Manihot esculenta Cranz, Oct 2019, S. S. Nascimento, (ARM651).
Notes
Concatenated gene analysis clustered Nigrospora pernambucoensis in a well-supported clade, sister to N. endophytica sp. nov (Fig. 1). Nigrospora pernambucoensis has highly branched hyphae with a smaller diameter than N. endophytica. More than one shape of conidiogenous cells (globose and obpyriform) can be observed in N. pernambucoensis, while N. endophytica only globose shape were visualized (Fig. 4). Hyaline vesicles are present in N. pernambucoensis and absent in N. endophytica. Conidia can be black in color in addition to pale brown to dark brown, and are larger in N. pernambucoensis with 12.5–20 µm diam vs pale to dark brown conidia with 10–17.5 µm diam in N. endophytica.
Nigrospora pyriformis M. Wang & L. Cai, Persoonia 39: 136 (2017).
Description of the species
Wang et al (2017).
Type
CHINA, Jiangxi Province, on Citrus reticulate, 11 Mar 2012, X. M. Tan, Holotype (HMAS 247067), Ex-type culture (CGMCC3.18122 = LC2045).
Material examined
BRAZIL, Pernambuco state, Condado municipality, in cassava field, 7° 35′ 18.959″ S 35° 4′59.022″W, alt. 118 m, isolated as an endophyte from stem of Manihot esculenta Cranz, Jan 2020, A.C.Q. Brito, (ARM970).
Notes
Nigrospora pyriformis has no host specificity, having already been isolated in Camellia sinensis, Castanopsis sp., Chenopodium album, Citrus sinensis, Lindera aggregata, Musa paradisiaca, Rosa sp., Rubus reflexus, and Saccharum officinarum (Wang et al. 2017; Raza et al. 2019; Chen et al. 2020). However, until the present study its distribution was restricted to China, and related to plant diseases, such as leaf spot (Chen et al. 2020). In our study, one strain of N. pyriformis was identified inhabit healthy stem of Manihot esculenta Cranz. It is a new host record for this species in the world. Besides that, this is the first occurrence of N. pyriformis outside of China.
Nigrospora vesicularifera M. Raza & L. Cai, Fungal Diversity 99: 96 (2019)
Description of the species
Raza et al (2019).
Type
CHINA, Guangdong Province, Zhanjiang City, Leizhou County, on Saccharum officinarum, Aug 2016, M. Raza and Y. Z. Diao, Holotype (HMAS 248077), Ex-holotype living culture (CGMCC 3.19333 = LC12052).
Material examined
BRAZIL, Pernambuco state, Condado municipality, in cassava field, 7° 35′ 18.959″ S 35° 4′59.022″W, alt. 118 m, isolated as an endophyte from stem of Manihot esculenta Cranz, Jan 2020, A.C.Q. Brito, (ARM975).
Notes
Nigrospora vesicularifera was introduced by Raza et al (2019) in a prospective study of pathogenic fungi associated with sugarcane diseases (Saccharum officinarum) in China. Since the description of the species no records of new hosts, new geographic occurrence or lifestyle (endophytic or pathogenic) quoting this species have been found. In the present study, N. vesicularifera was identified in a new host, Manihot esculenta Cranz, as endophytic in Brazil. This also represents a new geographic occurrence for this species outside Guangdong Province in China.