First Report of Bipolaris oryzae on Typha latifolia and the Pathogenicity of Its Isolates on Diﬀerent Rice Varieties

Rice ( Oryza sativa L.) is the staple food of more than half of the world population. However, its production is facing several biotic constraints. Among serious biotic factors that harm rice crops, the Helminthosporium disease has severe adverse impacts on rice yield, generating heavy losses of up to 90%. Four Bipolaris oryzae isolates were recovered for the first time from leaf lesions in the weed species Typha latifolia , and then subjected to pathogenicity tests on several rice varieties. e results indicated that Moroccan isolates of B. oryzae altered the leaf surface of five rice varieties tested. Among four isolates, Hor4 was the most pathogenic, showing high aggressiveness on the Cererrer and Elio varieties, with disease severity of 92.59%, followed by the Hor1, Hor2, and Hor3 isolates. e Arpa variety showed higher resistance to the Hor1 isolate, with a severity index of 35.18%. rough mycelial cutting or conidial suspension, B. oryzae isolated from T. latifolia was able to produce conidia on the leaves of this weed species.

According to Lage (1997), the presence of Pyricularia infection, Helminthosporium disease, and weeds (Echinochloa crus-galli, Panicums spp., Typha spp., and Cyperus spp.) could slow down rice production (Boulet & Bouhache, 1990). e Food and Agriculture Organization of the United Nations (FAO) stated in 2003 that the most common weed species affecting rice in the Mediterranean region belong to Poaceae and Cyperaceae. In the Gharb region, the most common weeds are Panicum (P. repens, Ligustrum obtusifolium Del.), Typha (T. latifolia L., T. marsii Bat.), Scirpus spp., Cyperus spp., and Echinochloa spp. (Miège, 1951). ese species are well adapted to the different agroecosystems where rice is cultivated and can promote the conservation and multiplication of pathogenic species (Pugh & Mulder, 1971;Singh et al., 2008). Benkirane et al. (2000) observed that Moroccan isolates of Pyricularia oryzae, originating from Stenotaphrum secundatum, are pathogenic on rice. Likewise, Serghat, Mradmi, et al. (2005) found that the fungal pathogen Pyricularia oryzae, isolated from Echinochloa phyllopogon and Phragmites australis, induced leaf lesions and sporulate on the foliage of certain rice varieties. e leaves of Typha latifolia, a perennial plant species found around rice fields, oen show similar leaf lesions to those observed in rice plants infected with B. oryzae. In this study, B. oryzae isolates obtained from leaf lesions in T. latifolia were subjected to pathogenicity tests on the leaves of five rice varieties. Indeed, the objective of this study was to highlight the role of an infectious reservoir, such as T. latifolia, in harboring and spreading leaf pathogens, particularly B. oryzae, to neighboring rice fields, which will cause significant damage to the rice fields.

Mycological Analysis
Infected T. latifolia plants showing leaf symptoms were transferred to the laboratory for microscopic examination, isolation, purification, and pathogenicity test. e blotter method was used for the isolation of B. oryzae. Infected leaf tissues of T. latifolia were collected, cut into small pieces, sterilized by immersion in 0.5% sodium hypochlorite for 1-5 min, and then washed three times with sterile distilled water. e leaf fragments (1 cm in diameter) were placed in sterile Petri dishes containing two discs of filter paper moistened with sterile distilled water. e dishes were then incubated at 22°C in alternating 12-hr light and darkness. Aer 48 hr, the leaf fragments were examined under an optical microscope at magnification ×100 for observation of the presence of fungal spores. e fungal species was determined using identification keys (Ou, 1985;Tarr, 1962).
Bipolaris spores were then single-spored with a capillary tube, placed on agar medium (agar-agar: 15 g, distilled water: 1,000 mL), and subsequently transferred using a sterilized needle to the surface of a rice flour-based medium (rice flour: 14 g, agar-agar: 15 g, yeast extract: 4 g, distilled water: 1,000 mL). Four isolates of B. oryzae were cultured.

Pathogenicity Test
e seeds of five rice varieties, namely, Elio, Taibonet, Arpa, Eurano, and Cererrer, were disinfected by immersion in 5% hypochlorite sodium solution for 2 min, followed by three rinses with sterile distilled water. ey were then dried on a sterile filter paper and pre-germinated on Petri dishes 90 mm in diameter containing sterile cotton soaked with sterile distilled water. Aer 72 hr of incubation in the dark at 28°C, the obtained plantlets were transplanted into pots filled with Mamora soil and then placed in a greenhouse. e seedlings were watered with tap water until they reached the stage required for inoculation, that is, when they had grown four to five true leaves.

Inoculum Preparation
Inoculum was prepared by independently growing each of the four isolates of B. oryzae (Hor1, Hor2, Hor3, Hor4) on a rice flour-based medium (rice flour: 15 g; Agar-agar: 15 g; yeast extract: 4 g and 1,000 mL of distilled water), which is favorable for the sporulation of B. oryzae, for 15 days (continuous photoperiod, 25°C). Aer incubation, the cultures were flooded with 15 mL of distilled water, and spores were dislodged using a sterile spreader.
Aerward, the fungal suspension was filtered through a fine mesh cloth to separate the spores from the mycelial fragments. e concentration of the conidia was adjusted to 10 5 conidia/mL using Malassez slide by adding sterile distilled water supplemented with a drop of Tween 20 and 0.5% gelatin.

Inoculation of Typha latifolia With Spore Suspension
Healthy leaves were soaked in the conidial suspension of each B. oryzae isolate. Control leaves were soaked in distilled water containing a drop of Tween 20 and gelatin. e leaves were placed in 9-cm-diameter Petri dishes containing glass beads moistened with sterile distilled water and then incubated under continuous white light at room temperature for 7 days.

Inoculation of Typha latifolia With Mycelial Discs
Healthy leaves were placed in 9-cm-diameter Petri dishes containing glass beads in the presence of sterile distilled water. ey were then inoculated with the mycelial plug (5 mm in diameter) of each isolate: one mycelial plug was placed on the central part of leaf segment, and another mycelial plug was deposited near the leaf apex. Noninoculated leaves (treated with water agar discs only) served as a control. In both detached leaf assays, controls were treated with sterile distilled water containing 0.01% Tween 20. Inoculated and control leaves were kept at ambient temperature under a natural light/dark cycle in the laboratory.

Inoculation of Rice Varieties
Rice seedlings at the stage of five to six leaves were inoculated by foliar spraying of 60 mL of spore suspension at 10 5 conidia/mL concentration using a hand compressed spray. Control plants were sprayed with sterile distilled water containing Tween 20 and gelatin. Aer spraying, all plants were covered with a black plastic bag and placed in a greenhouse for the development of symptoms. e plastic bag was used for the first 48 hr to ensure 100% relative humidity during conidium germination and fungal penetration. e replication consisted of three pots with three plants per pot for each rice variety. e experiment was repeated three times. Acta Mycologica / 2022 / Volume 57 / Article 573 Publisher: Polish Botanical Society 2.8. Assessment of Infection Severity e degree of leaf necrosis was evaluated on the seventh day aer inoculation for the four rice varieties artificially inoculated with conidial suspension, and at 2 days later for the rice inoculated with mycelial plugs. Disease severity was assessed as the proportion of infected leaf area on randomly selected rice plants. It was estimated using a disease rating scale of 1-9, as suggested by Notteghem et al. (1980), on the last two leaves of each infected rice plant. e results are described in Table 1. Table 1 Disease rating scale (Notteghem et al., 1980).

Note
Diseased leaf area (%) For analysis, the severity scale was converted into Percentage Severity Index (IS) using the following formula: where x i : disease severity scale; n i : number of infected plants (or leaves) with a rating of i; N t : total number of plants observed; 9: maximum disease severity scale.

Sporulation on the Host Plant
Sporulation was determined according to the technique of Hill and Nelson (1983) by estimating the average number of conidia produced per unit area of the infected leaves (expressed in number of spores/cm 2 ).
At 7 days aer inoculation, rice leaves that showed lesions were removed, cut into four-five fragments, and then placed in Petri dishes containing filter paper moistened with sterile distilled water (one sheet per dish). e dishes were placed under continuous fluorescent light for 72 hr at 28°C.
Each leaf segment was then collected in a test tube containing 1 mL of sterile distilled water. Aer that, the tubes were agitated in a vortex mixer for 2 min to detach the conidia from the mycelium. e conidia of the pathogen were counted using a Malassez slide under an optical microscope, with 10 counting for each sample.

Statistical Analysis
e statistical analysis of data was conducted using analysis of variance aer transformation of percentages to arcsin √P. Comparison between means was performed using the least significant difference (LSD) test at p < 0.05.

Morphological Characteristics
On the rice flour-based medium, fungus isolates formed fluffy and cottony aerial mycelia. e colony was gray to dark greenish gray in color ( Figure 1);  the conidiophores grew in singles or in groups, branched or simple, multiseptate, flexuous, sometimes with geniculate upper part, brown to black. e conidia of the fungal species were straight, cylindrical, usually curved, light brown to golden brown, with six to 14 transverse partition. e conidial size ranged from 63 to 153 (avg. 109) μm × 14 to 22 (avg. 17) μm ( Figure 1B). Morphologically, this fungus was therefore identified as B. oryzae (Ellis, 1971;Ou, 1985;Tarr, 1962).  Figure 2).  Table 3 shows that B. oryzae isolates Hor4 and Hor2 induced fairly large lesions (20.8 and 16.7 mm, respectively) on leaves of T. latifolia, compared to those induced by Hor3 (11 mm) and Hor1 (9.2 mm) (Figure 3).

Sporulation of Bipolaris oryzae Isolates
rough inoculation with either mycelial discs or spore suspensions, B. oryzae was able to sporulate on T. latifolia leaves, and no significant difference in intensity between the isolates tested was observed. Indeed, the average number of conidia ranged between 0.13 × 10 5 conidia/cm 2 and 0.83 × 10 5 conidia/cm 2 for the first technique (Table 4), and from 0.1 × 10 5 conidia/cm 2 to 0.6 × 10 5 conidia/cm 2 for the second technique (Table 5).

Inoculation of Rice Varieties With Conidial Suspension
Aer 7 days of inoculation, all the rice varieties tested displayed high sensitivity towards the four isolates of B. oryzae studied, as reflected by necrotic areas on the inoculated leaf surfaces. us, similar symptoms were observed in the five varieties of rice; they were generally of reddish-brown color, oval shape surrounded by a pale yellow halo, and were relatively uniform and regularly distributed (Figure 4). As shown in Table 6, among the five rice varieties tested, Elio was the most sensitive to the Hor1 isolate, with a severity index of 90.73%, followed by the varieties Taibonet, Cererrer, and Eurano, whose severity indexes reached 85.18%, 79.63%, and 79.62%, respectively. In comparison, the Arpa variety was less sensitive, showing a severity index of 35.18% against the same isolate.  However, the severity varied between 87.03% and 59.26% for the varieties Taibonet, Eurano, and Arpa. Table 7 shows that for a given isolate, the severity varies depending on the inoculated rice variety. e Hor4 isolate was found to be most pathogenic to the Cererrer and Elio rice varieties, whose disease severity index reached 92.59%, followed by the Taibone (87.03%) and Eurano (84.25%) varieties.
e Hor3 isolate was highly pathogenic to the Cererrer variety (90.73%) and less pathogenic on Elio and Eurano varieties, with respective disease indexes of 79.63% and 74.07%. e severity indexes for the Hor2 isolate were the highest in the Cererrer and Elio rice varieties (90.74% and 81.74%, respectively), but did not exceed 74.99% in the Eurano, Taibonet, and Arpa varieties. Hor1 was more pathogenic to the Elio and Taibonet varieties, with respective indexes of 90.73% and 85.18%.
e sporulation ability of B. oryzae isolates from T. latifolia on the leaves of five varieties of rice showed pronounced variability between isolates. e highest spore number of the Hor1 isolate was observed in the Eurano variety, with 1.16 × 10 5 spores/cm 2 , followed by the Arpa and Taibonet varieties, with 0.26 × 10 5 and 0.16 × 10 5 spores/cm 2 , respectively. However, the sporulation was very low in the Elio and Cerrerer varieties, in which the number of spores was reduced to 0.06 × 10 5 spores/cm 2 (Table 8).
e sporulation intensity of the Hor3 isolate on the leaves of the Arpa and Cererrer varieties was high (1.43 × 10 5 and 1.33 × 10 5 spores/cm 2 , respectively). In comparison, on the leaves of the Eurano and Elio varieties, the spore number was 0.7 × 10 5 and 0.63 × 10 5 spores/cm 2 , respectively. Moreover, no spore was found on the leaves of the Taibonet variety (Table 8). e maximum spore production of the Hor4 isolate was observed on the leaves of the Eurano variety, with 2.26 × 10 5 spores/cm 2 . However, no spore was found in the other varieties (Taibonet, Arpa, Cererrer, and Elio).

Discussion
In the pathogenicity tests, Moroccan isolates of B. oryzae, which were isolated for the first time from the leaves of T. latifolia, were revealed to be pathogenic on rice seedlings, as indicated by necroses and discoloration on a large portion of the leaf blades of the five studied varieties. We can consider T. latifolia an alternative host of B. oryzae. Our result is in agreement with previous research results indicating that B. oryzae had a wide range of hosts, including Oryza sativa, Triticum aestivum, Panicum virgatum, Zea mays, Brachypodium distachyon (Farr & Rossman, 2017;Kaspary et al., 2018;Manamgoda et al., 2014), and Typha orientalis (Wang et al., 2019). Based on the severity index, B. oryzae isolates showed variable pathogenic capacity to the five tested rice varieties. e high pathogenicity of B. oryzae on rice varieties is supported by Monira et al. (2021), who reported that B. oryzae can attack rice seedlings and seeds and can remain viable for up to 5 years on Heera 2 hybrid rice seeds under suitable storage conditions. Based on their response to B. oryzae inoculum, the tested varieties were classified as moderately sensitive to highly sensitive according to the severity scores defined by Boka et al. (2018).
Regarding the sporulation ability, the tested isolates of B. oryzae succeeded in producing abundant spores on the leaf lesions, suggesting successful infectivity irrespective of rice variety and host. e attack of a plant by a pathogen depends on the pathogen itself (Notteghem et al., 1980) or on the host plant genotype (Marchetti & Bonman, 1989). However, the severity scores can vary depending on the isolates and rice variety. Similar observation was made by Ouazzani Touhami et al. (2000), who found different pathogenicity levels among isolates of Helminthosporium spiciferum and H. australiensis, whose capacity to sporulate on host plants also depends on the rice variety.
Helminthosporium can be isolated from a wide range of hosts, including corn and grasses (Nelson & Kline, 1961, 1962Nelson et al., 1963). e parasitism of B. oryzae on corn was reported by Ou (1972) and Vidhyasekaran et al. (1986). e lesions appeared at approximately 18 hr aer the inoculation of B. oryzae on the rice plant (Dallagnol et al., 2009). e results showed that the pathogenicity of B. oryzae was not specific to the rice from which it was isolated. is fungus can, without doubt, extend to other species widely cultivated in the vicinity of rice fields.  have, in fact, shown that B. oryzae can be isolated from grasses, such as wheat, corn, and barley, that are located close to rice fields. e fungi present on leaf lesions in weeds can constitute a potential source of inoculum for rice plants. e presence of these weeds in and around rice fields helps maintain a high level of contaminating inoculum, promoting the progression of the epidemic. Indeed, the production of secondary inoculum via multiplication of infectious elements on the leaf lesions generates new contaminations and allows the disease to progress in rice fields (Serghat, Mradmi, et al., 2005;. According to Boulet and Bouhache (1990), the presence of an adventitious flora adapted to the conditions of rice fields, such as Echinochloa crus-galli and E. phyllopogon, greatly compromises the health of rice fields. Likewise, this flora harbors the same fungi that are found on rice leaf lesions. In the same context, the studied mycoflora on Echinochloa phyllopogon and Phragmites australis (two weeds adapted to rice fields) showed two types of fungi: true rice pathogens (Pyricularia grisea, Helminthosporium oryzae, H. sativum, H. australiensis, H. spiciferum, and Curvularia lunata) and saprophytes that cause rice discoloration (Trichoderma harzianum, Alternaria alternata, Nigrospora oryzae, Epicoccum nigrum, Fusarium moniliforme, Cladosporium herbarumand, and Trichothecium roseum) (Serghat, Mradmi, et al., 2005). Weeds in rice fields can also harbor other pests, including viruses, bacteria, and insects. us, in the presence of Typha sp., the development of Sesamia (Sesamia nonagrioides) in rice fields is much faster because this weed species is a plant host for the first larval stages of this predatory rice insect (Fazeli, 1992). Echinochloa crus-galli was identified as capable of hosting and transmitting the southern rice black-streaked dwarf virus in South China (Li et al., 2012). In addition, E. crus-galli has proven to be an important reservoir of Aphid and Barley yellow dwarf luteovirus (BYDV) (Geissler & Karl, 1989). According to Bouhache et al. (1989), weeding rice fields and eradicating the surrounding weeds may protect rice plants from infection by the inoculum produced on these weeds. Our data provide important information on the novel isolates of B. oryzae and on the possibility of latent inoculum transmission that leads to disease in crops and weeds surrounding rice fields. However, the development of pathogen control strategies based on the genetic structure of the pathogen populations would certainly be effective (Nagaty & El Assal, 2011), and more knowledge is needed on host shiing and host expansion in fungal plant pathogens.