Physiological characterization of Cochliobolus bicolor (sin. Bipolaris bicolor) from wheat BRS 264 seeds

Caracterização fisiológica de Cochliobolus bicolor (sin. Bipolaris bicolor) de sementes de trigo BRS 264

Abstract

Several diseases cause a reduction on the wheat (Triticum aestivum L.) yield, among which is helminthosporiosis, which infects the plant in all its stages of development. The objective of this work was to carry out the physiological characterization, under different temperature regimes of Cochliobolus bicolor isolates from BRS 264 wheat seeds. The six isolates of C. bicolor were recovered from the mycoteca stored in the Plant Pathology Laboratory of the Goiás State University. For the physiological evaluation, C. bicolor isolates were inoculated in Petri dishes containing PDA medium and subjected to six temperatures: 10°, 15°, 20°, 25°, 30° and 35°C. The evaluations were carried out obtaining the measurements of mycelial growth during 5 days. At 8 days after inoculation (DAI), a total of 10 mL of sterilized distilled water was added to each Petri dish, followed by the release of spores with a Drigalsky loop, the suspensions obtained were measured in a Neubauer chamber. For the germination tests, plastic Petri dishes containing agar-water medium were used, to which 100 µL C. bicolor spores suspension were added. Then the plates were taken to BOD at 25°C and constant light. The germ tube length of five spores per Petri dish was measured during five hours. The experimental design was completely randomized, with five replications (Petri dishes) for each C. Bicolor isolate. The temperature of 25°C provided greater mycelial growth area, sporulation and spore germination.

Keywords:
Triticum aestivum; mycology; helminthosporiosis; epidemiology

Resumo

Diversas doenças causam redução na produtividade do trigo (Triticum aestivum L.), dentre elas a helmintosporiose, que infecta a planta em todos os seus estágios de desenvolvimento. O objetivo deste trabalho foi realizar a caracterização fisiológica, sob diferentes regimes de temperatura, de isolados de Cochliobolus bicolor provenientes de sementes de trigo BRS 264. Os seis isolados de C. bicolor foram recuperados da micoteca armazenada no Laboratório de Fitopatologia da Universidade Estadual de Goiás. Para a avaliação fisiológica, isolados de C. bicolor foram inoculados em placas de Petri contendo meio BDA e submetidos a seis temperaturas: 10°, 15°, 20°, 25°, 30° e 35°C. As avaliações foram realizadas obtendo-se as medidas de crescimento micelial durante 5 dias. Aos 8 dias após a inoculação (DAI), adicionou-se um total de 10 mL de água destilada esterilizada em cada placa de Petri, seguido da liberação dos esporos com alça de Drigalsky, as suspensões obtidas foram mensuradas em câmara de Neubauer. Para os testes de germinação conidial, foram utilizadas placas de Petri plásticas contendo meio ágar-água, às quais foram adicionados 100 µL de suspensão de esporos de C. bicolor. Em seguida, as placas foram levadas à BOD e submetidas a 25°C e luz constante. O comprimento do tubo germinativo de cinco esporos por placa de Petri foi medido durante cinco horas de germinação. O delineamento experimental foi inteiramente casualizado, com cinco repetições (placas de Petri) para cada isolado de C. bicolor. A temperatura de 25°C proporcionou maior área de crescimento micelial, esporulação e germinação de esporos.

Palavras-chave:
Triticum aestivum; micologia; helmintosporiose; epidemiologia

1. Introduction

They are found approximately 70 host species in the world of Species of the genus Bipolaris (Farr and Rossman, 2020), surviving on seedlings or saprophytically on seeds, also occurring in high incidence in barley, wheat and other grasses, causing helminthosporiosis or foliar spot disease, infecting the plants at all stages of development (Kobayasti and Pires, 2011). The absorption of nutrients and water is affected when the fungus attacks underground organs, causing wheat foot rot. When the pathogen attacks the plant's green organs, it compromises photosynthetic processes, so called brown spot disease (Farr and Rossman, 2020). The conditions of high temperature and humidity promote the germination of conidia, thus causing the appearance of fungi that will consequently harm the plant's development, affecting seed germination. So that temperate and tropical climate and humid regions favor the Bipolaris spot disease, causing high production losses (Iftikhar et al., 2009). Within this context, it is important to emphasize that fungi are essential biological components of ecosystems, performing diverse roles that ensure habitat balance and socioeconomic stability (Calaça et al., 2022).

The pathosystem requires epidemiological and diagnostic studies, since artificial inoculations are required and, for this purpose, the mass production of pathogen inoculum in the laboratory (Marcuzzo and Xavier, 2017). However, there are several obstacles to developing studies on the pathosystem, which presents different responses to temperature, then there is difference as for the physiology between isolates even being of the same species (Angelotti et al., 2014; Shristi et al., 2018). Thus, laboratory research seeks better knowledge of the pathosystem, using methods for determining thermal thresholds and the best temperature for mycelial development and maximum sporulation of the pathogen in vitro, where they are postulated in several epidemiological studies and tests of control methods of the disease (Pereira et al., 2016; Muniz et al., 2025). However, studies of mycelial growth and in vitro sporulation have been growing a lot due to the knowledge of the physiological characters of fungi (Hendges and Nozaki, 2017).

Temperature can interfere with mycelial growth, colony color, sporulation and conidia germination, requiring an adequate temperature for the pathogen to complete its cycle (Camera and Deuner, 2017). However, high temperatures affect metabolic activity and enzyme-mediated synthesis, where high temperatures will cause an increase in the rate of the metabolic reaction, consequently denaturing the enzymes. Fungi commonly develop between 0ºC and 45ºC, not forgetting the fungi that have the capacity to adapt to extreme temperatures (Maia et al., 2015). The temperature can affect fungi germination, in order to regulate the speed of its germination, and thus, favoring disease epidemics. These climate changes can contribute to great impacts on phytosanitary problems, thus, understanding the pathogen's development when submitted to the environmental temperature variations can promote a strategy to disease control (Maia et al., 2011). Characterization is important to identify and know the variability between isolates of a particular pathogen. Thus, being able to develop adequate control methods. The objective of this work was to carry out the physiological characterization of Cochliobolus bicolor isolates from BRS 264 wheat seeds when subjected to different temperature regimes.

2. Material and Methods

2.1. Obtaining Cochliobolus bicolor (sin. Bipolaris bicolor) isolates

Cochliobolus bicolor fungus were obtained from individual seeds of wheat BRS 264. The mycelium grown on the seeds were peaked to Petri dishes containing Potato Dextrose Agar (PDA) medium, added with antibiotic Amoxicillin (250mg / 250mL of PDA), were incubated into BOD at 25ºC for 7 days and 12 h photoperiod. After 7 days at 25ºC and 12 h photoperiod, the obtained fungal colonies were subjected to new subculture in PDA medium until colony purification. Thus, six isolates of Cochliobolus bicolor were obtained: F-24-01, F-24-02, F-24-03, F-24-04, F-24-05 and F-24-06.

The purified isolates were preserved in PDA medium at a temperature of 5°C by the Castellani method. These isolates are part of the Phytopathogenic Fungi Collection of the Phytopathology Laboratory of UEG, Ipameri Unit. The isolates were reactivated for subsequent experiments, in PDA medium, from samples kept at low temperature in a freezer at the Laboratory of Phytopathology at UEG. The purified isolates were preserved at 4°C by the Castellani method in the mycoteca at the UEG Plant Pathology Laboratory. DNA isolation, amplification and molecular analyses were made by Muniz et al. (2024a).

2.2. Mycelial growth and sporulation under different temperature regimes

Agar plugs (7 mm Ø) containing mycelium from C. bicolor isolates were removed from ten-day-old colonies and transferred to the center of Petri dishes (85 mm Ø) containing PDA medium. Then, the plates were transferred to BOD at 10ºC, 15ºC, 20ºC, 25ºC, 30ºC and 35ºC and 12 h photoperiod using fluorescent lamps of 20W, 75RS (Philips® brand) for 5 days. The radial growth of the mycelium was evaluated at 1, 2, 3, 4 and 5 days after inoculation (DAI), from the average of two diametrically opposite diameters (Carvalho et al., 2008).

After the last reading of radial mycelial growth at 5 DAI, another three days were waited, completing 8 DAI, to quantify the spore production by the C. bicolor isolates at the six evaluated temperatures. For that, a total of 10 mL of sterilized distilled water (SDW) was added to each Petri dish, followed by the release of the spores with a Drigalsky strap. Then, the spores were collected in becker and filtered through sterile gauze. The concentrations of the obtained suspensions were measured in a Neubauer chamber, counting the spores in five quadrants of the chamber for each Petri dish (Carvalho et al., 2008). The experimental design was completely randomized, with five replications (Petri dishes) for each of C. bicolor isolate.

2.3. Germination of spores on water agar medium under different temperature regimes

A total of 100 µL of the C. bicolor spore suspension (calibrated at 1.6 x 10⁵ conidia mL^-1) was spread´, with aid of a Drigalsky loop, on the surface of the water agar (WA) culture medium, which was contained within plastic Petri dishes (60 mm Ø). Then, the plates (four plates per C. bicolor isolate) were transferred to BOD, where did stay at 25°C and constant light, where germination occurred. The germination of the spores was verified by placing the plates under a light microscope to visualize and measure the length of the germ tube of five spores per Petri dish every one (01) hour, during five hours, using the LAS-EZ program to perform the measurements.

2.4. Statistical analysis

The results concerning mycelial growth and sporulation under different temperature regimes were submitted to analysis of variance, Scott-Knott test (P<0.05) by prefixing the last reading date of radial growth and sporulation. Besides, regression analysis for obtaining significant models for mycelial growth, sporulation and spore germination were performed in the statistical program Sisvar 5.3 (Ferreira, 2011).

3. Results

3.1. Mycelial growth under different temperature regimes

The mycelial growth of C. bicolor was determined by the colony area (cm²) varying with temperature (ºC) and over time. The optimal growth, estimated through the derivative regression equations, was 24°C (Figure 1), which it was the temperature that provided the maximum mycelial growth (Table 1). Regression equations were fitted by a second degree polynomial model, with a high coefficient of determination (R²) and significant at 1%. The coefficient of determination (R²) allowed to infer that the temperature variation influences between 61-74% mycelial growth.

Figure 1
Mycelial growth of Cochliobolus bicolor (at 5th DAI) as a function of temperatures at 10ºC, 15ºC, 20ºC, 25ºC, 30ºC and 35ºC. Ipameri, Goiás, Brazil, 2021.

Table 1
Regression model for mycelial growth of Cochliobolus bicolor as a function of temperatures at 10ºC, 15ºC, 20ºC, 25ºC, 30ºC and 35ºC. Ipameri, Goiás, Brazil, 2021.

Isolate	Model(1)	R2	P(≤X)	TMMG(2)
F-24-01	Y = - 0.278927x2 + 13.359367x – 115.441457	0.71	0.01	22° C
F-24-02	Y = - 0.283187x2 + 13.539867x – 115.727457	0.71	0.01	24° C
F-24-03	Y = - 0.281250x2 + 13.578421x – 116.901857	0.66	0.01	24° C
F-24-04	Y = - 0.258704x2 + 12.568870x – 109.237343	0.61	0.01	24° C
F-24-05	Y = - 0.251203x2 + 12.036837x – 101.990514	0.69	0.01	24° C
F-24-06	Y = - 0.296219x2 + 14.127801x – 120.150943	0.74	0.01	24° C

• ⁽¹⁾The models for each isolate correspond to the regression curves shown in Figure 1.
• ⁽²⁾Temperature of maximum mycelial growth, obtained from the model derivative.

3.2. Sporulation under different temperature regimes

Sporulation started from the 8th DAI. However, the isolates did not produce conidia at 10ºC, 15ºC, 20ºC, 30ºC and 35ºC, under 12 h photoperiod, that is, only at 25ºC that conidia was produced. Isolate F-24-04 was superior to the others as for sporulation at 25ºC (Table 2).

Table 2
Sporulation of Cochliobolus bicolor (5th DAI) at 25ºC. Ipameri, Goiás, Brazil, 2021.

Isolate	Sporulation at 25° C (conidia ml-1)
F-24-01	0.53 x 105 c
F-24-02	0.85 x 105 c
F-24-03	0.50 x 105 c
F-24-04	3.57 x 105 a
F-24-05	0.64 x 105 c
F-24-06	1.86 x 105 b
Mean	1.37 x 105
CV (%)	36.01

• Means followed by the same letters do not differ statistically from each other, according to the Scott-Knott test (P≤0.05).

3.3. Germination of spores on water agar medium under different temperature regimes

The germination of C. bicolor conidia, obtained from plates at 8 DAI, was evaluated at 25ºC and constant photoperiod, that is, the best temperature for mycelial growth and the only in which there was sporulation. Germination occurred in the first hours and progressed until the length of the germ tube reached a size equal to twice the largest dimension of the spore body, an event that occurred at 5 hours after inoculation (Figure 2F). Through regression analysis, significant linear models were obtained at P≤0.05, with a high coefficient of determination (R²), where the temperature of 25°C influenced between 86-96% the growth of the germ tube (Table 3).

Figure 2
Conidia of C. bicolor incubated at 25°C and constant light, in WA medium, showing germ tube growth as a function of time. A. 0 h of incubation, no germination (F-24-02); B. 1 h of incubation, showing unipolar germination (F-24-02); C. 2 h of incubation, bipolar germination (F-24-01); D. 3 h of incubation, unipolar (F-24-06); E. 4 h of incubation, bipolar (F-24-04); F. 5 h of incubation, unipolar (F-24-06). Bars: A (50 µm), B (45 µm), C and E (40 µm), D (35 µm) and F (60 µm).

Table 3
Regression model for Cochliobolus bicolor germination as a function of time (1 to 5 hours) and the length of the germ tube at 25ºC after 5 hours of inoculation on WA medium and constant photoperiod. Ipameri, Goiás, Brazil, 2021.

Isolate	Model	R2	P(≤X)	Germ tube length (µm)
F-24-01	Y = 19.209200x – 7.636000	0.96	0.05	91.7(ns)
F-24-02	Y = 14.577200x + 8.723600	0.75	0.05	77.1
F-24-03	Y = 24.402400x – 23.545200	0.93	0.05	106.3
F-24-04	Y = 21.724600x – 12.706600	0.86	0.05	112.0
F-24-05	Y = 16.325000x + 3.859400	0.96	0.05	86.1
F-24-06	Y – 23.763600x – 8.891200	0.96	0.05	115.1
Mean	-	-	-	98.0
CV (%)	-	-	-	23.06

• ^(ns)Not significant according to Scott-Knott test (P≤0.05).

4. Discussion

In the present work, mycelial growth showed a response up to 30°C, with the optimal growth obtained at 24°C. Similarly, for B. sorokiniana, the fungus responded up to 30°C with an optimal temperature of 23°C when observing the area of lesions in wheat plants (Prates and Fernandes, 2011). For most organisms, temperature, as well as light, is an environmental physical factor that allows interfering with their development and physiological processes (Babitha et al., 2008). It is noteworthy that all isolates had great mycelial growth at 24°C, on what F-24-01 was the only one suitable at 22°C, this low difference is due to the isolates being obtained from the same seed cultivar. The finding of studies related to the temperature of optimal mycelial growth of pathogenic fungi has been carried out in order to observe the best installation of individuals along a temperature gradient (Maia et al., 2015). In most fungi, the reproductive and vegetative system is affected by an abiotic factor such as temperature (Muniz et al., 2024b). In general, diseases develop when the ideal temperature for the vegetative growth of phytopathogenic fungi is reached (Sharma and Ahir, 2018).

According to Dias et al. (2005), temperatures close to the borderline can cause a drastic decrease in the mycelial growth and, thus, subsidize knowledge in the management of plant diseases. It is worth mentioning that all evaluated C. bicolor isolates did not show adequate mycelial growth when subjected to 35°C. This fact shows that the increase in temperature inactivates enzymes with a resulting effect on metabolism, impacting mycelial growth (Pathak et al., 2014). Considering the six C. bicolor isolates, sporulation was possible at 8 DAI. Such sporulation time seems to corroborate with other helminthosporiosis complex fungi. The fungus Drechslera tritici-repentis obtained sporulation on the 5th day of incubation at a temperature of 25°C (Tonin et al., 2014). While for the fungus Exserohilum turcicum, germination started after the 10th day of incubation at 25°C (Camera and Deuner, 2017).

Cochliobolus bicolor is a microorganism from temperate and hot and humid tropical regions, with specific conditions for its sporulation. Thus, in general, sporulation can be inhibited when found outside the optimal range of fungus growth. These may be explanations for the fact that C. bicolor did not sporulate along a temperature gradient. It is worth remembering that the effect of the temperature regime is not, in isolation, a factor for the in vitro C. bicolor conidiogenesis (Muniz et al., 2024b). However, in our work, temperature seems to have been the responsible factor to this event. Our results showed that there is an optimal temperature for mycelial growth and, simultaneously, sporulation of C. bicolor. Similarly, Rout et al. (2015) reported that the growth and maximum sporulation of A. alternata isolates were obtained at the same temperature of 30°C. F-24-04 was superior to the others in terms of sporulation at 25ºC. An explanation for this event lies in the fact that the interaction between isolate factors and temperature, both for mycelial growth and for sporulation, is indicative that they are intrinsic characteristics to each isolate, which react differently to temperature variations (Mello et al., 2018).

In works by Zadoks and Schein (1979), the spore is considered germinated when the length of its germ tube reaches a size equal to twice the largest dimension of the spore body, an event that occurred 5 hours after incubation of the C. bicolor spores. It is interesting to mention that this was a shorter time than found for other species of the Helminthosporiosis complex. As an example, we can mention the fungus Drechslera tritici-repentis, where the germ tube was detected after 2 hours and had a good development up to 9.5 hours after incubation (Tonin et al., 2014). At 25°C it was possible to obtain the maximum length of the germ tube for the fungus Exserohilum turcicum, which when subjected to 0°C, 5°C and 40°C, the conidia did not show non germination (Camera and Deuner, 2017). The germ tube length at 25ºC after 5 hours of inoculation on WA medium and constant photoperiod vary from 77.1 to 115.1 µm, however, without significant difference between the isolates. This range of values corroborate with Muniz et al. (2024a), whose values arrived until 90,8 µm when these isolated were submitted to the same conditions of temperature and light. Finally, the study of the reaction of fungi to physical factors is important for determining the temperature required by the pathogen for its growth, sporulation and germination of its conidia in the field, thus enabling the definition of the best time to prevent diseases in the field and minimizing the inoculum production (Alfenas and Mafia, 2016). Defining the optimal conditions for pathogen growth is critical because fungal diseases that affect plant systems remain major limiting factors for achieving high crop yields and ensuring economic sustainability (Silva et al., 2023).

5. Conclusion

The temperature for the maximum level of mycelial growth, sporulation and germination was 25°C.

Colonies of C. bicolor only sporulate at 8 DAI.

Temperature directly influences mycelial growth, spore production and germination of C. bicolor fungus, influencing between 61 to 74% the mycelial growth and 86 to 96% the germination.

Acknowledgements

The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for the scholarship to Juliana Oliveira Silva de Menezes. The authors would like to thank the State University of Goiás for the financial resources provided by the PrP/UEG Call n. 01/2024 Pró-Programas, Funding Term n. 76334379, SEI Process n. 202400020007877.

Data Availability Statement

The authors confirm that the data supporting the conclusions of this study are available within the article. There are no additional or underlying data that are not included.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  20 Mar 2026
• Date of issue
  2026

History

• Received
  23 Oct 2025
• Accepted
  03 Feb 2026