First Report of Fusarium Sheath Rot of Rice Caused by Fusarium incarnatum-equiseti Species Complex in the United States.

Fungal diseases, including sheath rot (Sarocladium oryzae), cause significant losses of yield and milling quality of rice (Oryza sativa). In August 2021, symptoms like sheath rot were observed on 20% of rice plants (cv. Presidio) in 1-hectare field in Eagle Lake, Texas. Initial lesions occurred on the upper flag leaf sheaths and were oblong or irregular oval, with gray to light brown centers, and a dark reddish-brown diffuse margin. Lesions enlarged, coalesced, and covered a large area of the sheath. Infection led to panicle rot with kernels turning dark brown. Unlike sheath rot, sheath infection also led to inside culm infection with irregular dark brown lesions. Infected tissue pieces were sterilized with 1% NaOCl for 2 min, followed by 75% ethanol for 30 s, washed in sterile H2O three times, air dried and incubated on PDA at 27℃. Fungal isolates were obtained from 15 diseased plant samples and their singled-spored fungal colonies were whitish, loosely floccose and produced light yellow pigmentation. On carnation leaf agar, macroconidia were slightly curved and tapered at the ends, with 3 to 5 septa, and measured 17.5 to 34.3 × 3.1 to 5.0 µm. Microconidia were ovoid, usually with 0 to 1 septum and were 4.0 to 15.5 × 2.5 to 4.5 µm. Spherical shaped chlamydospores were produced in chain. These morphological characteristics were consistent to those described for Fusarium incarnatum-equiseti species complex (O'Donnell et al. 2009), including F. incarnatum (Wang et al. 2021) and F. equiseti (Avila et al. 2019). For molecular identification, DNA of a representative isolate was extracted and ITS, LSU, and EF1 of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 2000), and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OL344049) was 99.61% identical to F. incarnatum-equiseti species complex (FD_01692) in Fusarium-ID database and 99.61% identical to F. equiseti (LC514690, KY523100, MW016539) and F. incarnatum (MH979697) in NCBI database. The LSU sequence (OK559512) was 98.77% similar to F. equiseti (MN877913, MN368509) and F. incarnatum (MH877332, MH877326); the EF1 sequence (OK570044) was 99.27% similar to F. equiseti (MK278902) in NCBI database. A phylogenetic analysis based on the concatenated nucleotide sequences grouped this isolate in the F. incarnatum-equiseti species complex clade at 100% bootstrap support. To evaluate pathogenicity, a conidial suspension of 1 x 106 conidia/ml or sterilized water (the controls) was injected into the sheaths and young panicles of three rice plants (cv. Presidio) at boot. Treated plants were maintained in a greenhouse at 25 to 30℃. After 3 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants but not on the controls. The same fungus was consistently re-isolated from the diseased plants. To our knowledge, this is the first report of Fusarium sheath rot caused by F. incarnatum-equiseti species complex in rice in the U. S. F. incarnatum-equiseti species complex has been reported to be associated with panicle infection in wild rice (O. latifolia) in Brazil (Tralamazza et al. 2021). F. incarnatum has also been reported to cause panicle rot in China (Wang et al. 2021). F. proliferatum has been reported to cause Fusarium sheath rot in India (Prabhukarthikeyan et al. 2021) and the U. S. (Cartwright et al. 1995). This research demonstrates the potential of different pathogens being involved in causing sheath rot of rice.

First Report of Leaf Spot of Rice Caused by Epicoccum sorghinum in the United States.

Brown spot (Cochliobolus miyabeanus), blast (Magnaporthe oryzae) and stackburn (Alternaria padwickii) are common diseases in rice with similar leaf spot symptoms. In August 2021, a leaf spot disease, with symptoms dissimilar to these diseases, occurred on almost 100% of the leaves and sheaths of rice plants (cv. Presidio) in a 1-hectare field in Eagle Lake, Texas. Lesions started as small dark brown spots on lower leaves and sheaths. The spots enlarged to become round or oval (1.5 to 5.0 mm) spots having round ends with gray centers, dark-brown borders or rings, and slight gold halos. The spots on the sheaths were similar to those on the leaf blades, with lesion size ranging from 2 to 5 mm. Pieces of infected tissue were cut from the margin of necrotic lesions, surface disinfected with 1% NaOCl for 2 min followed by 75% ethanol for 30 s and rinsed with sterile distilled water three times. The tissues were then dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 25℃ for 7 days. Two isolates (LS36 and LS37) were obtained, and their colonies were initially villose, gray at the center and pale at the margin, and then turned dark gray, with the reverse side becoming scarlet. Chlamydospores were unicellular or multicellular and massively produced in nearly spherical shape (11 to 26 × 10 to 22 µm, n=100). Pycnidia were dark and mostly spheroid (105 to 171 × 76 to 128 µm, n=100). Conidia were unicellular, hyaline, ellipsoidal, with the size of 3.6 to 5.8× 1.9 to 2.8 µm (n=100). These morphological characteristics were similar to those described for Epicoccum sorghinum (Zhou et al. 2018). The rDNA internal transcribed spacer (ITS), rRNA large subunit (LSU), and translation elongation factor 1 alpha (EF1) gene of an representative isolate (LS37) were amplified (Fell et al. 2000; Wang et al. 2014) and sequenced. The ITS sequence (OK189534) of the isolate was 96.95% identical to E. sorghinum (KX758542); the EF1 sequence (OK236518) was 98.37% identical to E. sorghinum (MN461167); and the LSU sequence (OK189535) was 99.29% identical to E. sorghinum (MK817520, MK817521, and MK817522). Rice plants (cv. Presidio) at heading were inoculated with the two isolates individually by placing a drop of conidial suspension of 1 x 106 conidia/ml or a 2-mm PDA plug of 7-day-old cultures on the wounded or unwounded leaves and sheaths (3 sites/leaf or sheath, 3 plants/treatment). The wound was made by penetrating the epidermis using a 0.5-mm-diameter pin. The plants inoculated with sterilized water or PDA-only plugs served as the controls. The treated plants were placed in a dew chamber at 26℃ for 2 days and then transferred in a greenhouse (25 to 30℃). After 5 days, typical symptoms, similar to those observed in the field, developed on all of the inoculated leaves and sheaths, with the wound inoculation inducing more rapid development of symptoms than the unwounded inoculation. No symptoms developed on the controls. The two isolates produced similar symptoms and the fungus was consistently re-isolated from the infected plants and confirmed to be E. sorghinum based on morphological characteristics. The pathogenicity test was repeated twice with similar results. To our knowledge, this is the first report of leaf spot caused by E. sorghinum in rice in the United States. This disease was first reported on rice in China in 2020 (Liu et al. 2020). This research will help identify this new disease from other leaf spot-like diseases and develop management strategies for control of this disease.

First Report of Brown Leaf Spot of Rice Caused by Curvularia hawaiiensis in the United States.

Multiple diseases, including brown spot (Cochliobolus miyabeanus), leaf spot (Epicoccum sorghimum), and blast (Magnaporthe oryzae), can cause spot-like symptoms on the leaves of rice. In July 2021, a disease showing symptoms like brown spot was observed in an 8-hectare field of rice, with disease incidence of >30%, in Beaumont, Texas. Lesions started as small pinhead-size blackish spots on leaf tips or from the edges of leaf blades. The spots enlarged to become irregular (most) or oval brown spots with a slight chlorotic halo. Diseased leaves were collected, washed in running tap water and cut into small pieces. Pieces of the tissue were surface sterilized with 1%NaOCl for 2 min followed by 75% ethanol for 30 s and then washed in sterile distilled water three times with each time lasting for 1 min. The disinfected tissue pieces were air dried, placed on potato dextrose agar (PDA) medium and incubated at 25℃. Initially fungal colonies were hairy in texture with light dark brown center and whitish edge and dark brown pigmentation at the reverse side. Mature colonies turned to black in the center and dark brown toward the edge, with black at the reverse side after 2 or more weeks of incubation. Conidia were oval to narrowly oblong, rounded at the ends, with 2 to 6 distoseptate, and 15 to 35 × 6 to 10 µm in size. These morphological characteristics were similar to those described for Curvularia hawaiiensis (Aslam et al. 2019; Ellis 1971; Kusai et al. 2015). For molecular identification, DNA was extracted and the two different rRNA regions internal transcribed spacer (ITS) and large subunit (LSU), and the two genes RNA Polymerase II (RPB1) and translation elongation factor 1 alpha (EF1) of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 200), and RPB1 and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OK397200) was 98.27% identical to C. hawaiiensis (KP131943); the EF1 sequence (OK492159) was 99.78% identical to C. hawaiiensis (KC503942); the LSU sequence (OK397295) was 98.96% identical to multiple C. hawaiiensis (MN055715, MH160813, MH875853, etc.); the RPB1 sequence (OK492160) was 97.41% identical to C. hawaiiensis (JN992363). To evaluate pathogenicity, three rice plants (cv. Presidio) at the 3-leaf stage were spray inoculated with a conidial suspension of 1 x 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 25 to 30℃. After 2 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants while no symptoms developed on the control plants. The same fungus was consistently re-isolated from the diseased plants. The pathogenicity test was conducted three times with similar results. To our knowledge, this is the first report of brown leaf spot caused by C. hawaiiensis in rice in the United States. Curvularia species are frequently associated with rice grain and cause blackish discoloration symptoms on grain kernels. Recently, however, C. hawaiiensis has also been reported to cause brown leaf spot in Malaysia (Kusai et al. 2015) and Pakistan (Aslam et al. 2019). This research will help identify this disease from other leaf spot-like diseases and develop effective management strategies.

Sterile White Basidiomycete Fungus Marasmius graminum: A New Pathogen Causing Seedling Blight in Rice.

In April 2018, damping-off of rice (Oryza sativa L.) seedlings at the 2-to-3-leaf stage was observed in three fields in the counties of Wharton and Matagorda of Texas and Jefferson-Davis Parish of Louisiana. All affected areas were 1 ha or greater, with 10 to 20% of the seedlings showing the symptoms. Infected seedlings showed dark-brown necrotic lesions on the roots and/or mesocotyls where white superficial mycelium was usually present. Symptomatic tissues excised from 10 diseased seedlings of each field were surface sterilized with 1% NaOCl, double rinsed in sterilized distilled water, and plated on potato dextrose agar (PDA). The plates were incubated at 25°C with a 12-h photoperiod in a growth chamber. After 48 h, hyphal tips of fungal colonies were transferred onto PDA and 12 isolates were obtained. Clamp connections and dolipore septa were observed in young hyphae, indicating that these isolates were a basidiomycete fungus. Young hyphal cells were binucleate based on safranin O stain (Bandoni 1979). No fruiting bodies or sclerotia produced on PDA after one month of incubation. Based on these morphological characteristics, these isolates were identified as belonging to sterile white basidiomycetes (SWB) (Howard et al. 1977). To further identify the isolates into the species level, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of a representative isolate was sequenced with primer ITS1 and ITS4 (Vinnere et al. 2005). The ITS sequence (GenBank acc. no. MT524457) had more than 97% sequence similarity with known Marasmius graminum strains from Denmark (JN943595) (Schoch et al. 2012) and Sweden (MH857692) (Vu et al. 2019). Pathogenicity was tested with three representative isolates in a growth chamber using a modified method (Carling and Leiner 1990). Pots (6.5 cm in diameter x 7.5 cm height) were filled with 100 g of sterilized sand and watered to field capacity. Five PDA plugs (4 mm in diameter) from 5-day-old growing culture were placed on the sand surface of each pot. Pots inoculated with PDA plugs without fungus served as the controls. Five seeds of rice cv. Presidio were planted into each pot and covered with 10 g of sterilized sand. Pots were maintained at 25±2°C in a growth chamber with a 12-h photoperiod for 14 days. There were four replicated pots for each treatment and the experiment repeated twice. After 2 weeks, severe damping-off and associated symptoms similar to those observed in the field appeared in the inoculated pots. No symptoms developed in the control pots. The same fungus was consistently re-isolated from infected plants. Based on morphological characteristics and rDNA-ITS sequencing, these isolates were identified as M. graminum. The SWB fungus was first reported as a causal agent of stem rot of snap bean in Florida (Howard et al. 1977) and Nebraska (Harveson 2002), root or hypocotyl rot of corn, snap bean, squash and peanut in Georgia (Sumner et al. 1979; Bell and Sumner 1984), and crown rot of pigeon pea (Cajanus cajan) in Puerto Rico (Kaiser et al. 1987). Later, the SWB strain (ATCC 28344) causing stem rot of snap bean in Florida was further identified as M. graminum based on nuclear large subunit rRNA gene (Vinnere et al. 2005). Comparing the ITS region of this isolate (AY445120) with our isolate revealed a 99% similarity. To our knowledge, this is the first report that the SWB fungus M. graminum causes seedling blight in rice. Identification of this new disease will help to develop management strategies for control of stand loss in rice.

Integration of Brassica Cover Crop with Host Resistance and Azoxystrobin for Management of Rice Sheath Blight.

Sheath blight caused by Rhizoctonia solani is the most important rice disease that can cause significant losses in grain yield and quality in the southern United States. Current management options for sheath blight primarily consist of fungicides, tolerant cultivars, and cultural practices. These options are not always very effective. Brassica plants have been used for soil fumigation to manage a variety of different soilborne pathogens. In this field study, the efficacy of a Brassica juncea cover crop integrated with use of a tolerant rice cultivar and fungicide application was evaluated in 2011, 2012, and 2013. The B. juncea cover crop significantly lowered sheath blight severity in all 3 years and led to a significantly higher grain yield in 2013 as compared with the fallow control. 'Presidio' rice had lower sheath blight severity and higher yield than 'Cocodrie' in 2012 and 2013. Fungicide applications with azoxystrobin at the label rate (0.16 kg a.i./ha) or half the label rate (0.08 kg a.i./ha) significantly reduced sheath blight severity in all 3 years, resulting in a yield increase in 2 of the 3 years. B. juncea along with use of a tolerant rice cultivar and half the label rate of azoxystrobin can be an effective approach for management of sheath blight in rice.