First Report of Passion Fruit Leaf Blight Caused by Nigrospora sphaerica in China

Abstract

Passion fruit (Passiflora edulis Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: ‘Panama Red’) newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50’49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1–13.5 μm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 μm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the β-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch’s postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 μL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: ‘Panama Red’). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%–75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.s Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: ‘Panama Red’) newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50’49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1–13.5 μm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 μm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the β-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch’s postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 μL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: ‘Panama Red’). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%–75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.

Plant Growth Promotion and Biocontrol of Leaf Blight Caused by Nigrospora sphaerica on Passion Fruit by Endophytic Bacillus subtilis Strain GUCC4

“…In the last decade, its market demand has consistently increased worldwide, including in China [ 12 ]. However, the cultivation and production of passion fruit, especially in low-temperature regions, including mountainous landscapes, is challenged by biotic damage, such as pests and phytopathogens [ 14 ]. Meanwhile, abiotic stresses, such as coldness and nutrient limitation, may negatively affect their growth [ 51 , 52 ].…”

“…are mainly considered plant endophytes [ 53 ]. Recently, N. sphaerica was reported to be the causal agent of leaf blight disease on different plants [ 15 , 17 ], including passion fruit [ 14 ]. To date, sustainable management strategies, including potential biological control resources, remain largely unexplored for controlling plant leaf blight caused by N. sphaerica .…”

“…The isolate N. sphaerica WYR007 from our previous study [ 14 ] was used in the experiments. The antagonistic activity of Bacillus spp.…”

“…However, due to the lack of local varieties and relatively low temperatures, plant pathogens are frequently reported to affect passion fruit production [ 7 , 13 ]. Leaf blight caused by N. sphaerica is a new and emerging disease on passion fruit grown in Guizhou [ 14 ]. Recently, Nigrospora spp., including N. sphaerica have been reported as causal agents for leaf blight disease over a broad spectrum of hosts (e.g., fruits, vegetables, and oilseeds) in East Asia, with incidence rates ranging from 2% to 90%, exhibiting significant strain–host specificity [ 15 , 16 , 17 ].…”

“…Recently, Nigrospora spp., including N. sphaerica have been reported as causal agents for leaf blight disease over a broad spectrum of hosts (e.g., fruits, vegetables, and oilseeds) in East Asia, with incidence rates ranging from 2% to 90%, exhibiting significant strain–host specificity [ 15 , 16 , 17 ]. For passion fruit leaf blight caused by N. sphaerica WYR007, the disease incidence was up to 70% [ 14 ]. The disease occurred on both young and old leaves, starting from the margins and then extending to the entire leaves.…”