Open AccessArticle

Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis

Adriany Pena de Souza

¹,

Juliana Fonseca Alves

¹,

Eliane Mayumi Inokuti

Fernando Garcia

¹,

Bruno Wesley Ferreira

Thaisa Ferreira da Nobrega

¹,

Robert Weingart Barreto

²,

Bruno Sérgio Vieira

^1,*

and

Camila Costa Moreira

Instituto de Ciências Agrárias, Universidade Federal de Uberlândia, Monte Carmelo 38500-000, MG, Brazil

Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa 36570-900, MG, Brazil

Koppert Do Brasil Holding Ltda, Piracicaba 13400-970, SP, Brazil

Author to whom correspondence should be addressed.

Agronomy 2024, 14(12), 2926; https://doi.org/10.3390/agronomy14122926

Submission received: 10 October 2024 / Revised: 17 November 2024 / Accepted: 19 November 2024 / Published: 7 December 2024

(This article belongs to the Special Issue Exploiting Beneficial Plant–Microorganism Interactions for Resilient Farming)

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Figure 1
Collection sites (red triangles) [IBGE (Instituto Brasileiro de Geografia e Estatística 2021). Design ilustration: Xavier, L. C. M. (2023)]. "> Figure 2
Sourgrass (Digitaria insularis) showing smut symptoms (growth reduction and formation of sori) on Anthracocystis panici-leucophaei in a field situation in Monte Carmelo, Minas Gerais (Brazil). "> Figure 3
Anthracocystis panici-leucophaei. (a,b) Sori on live plants (D. insularis). (c,d) Teliospores produced on fertile hyphae forming chains while immature (SEM). (e) Verruculose teliospores (SEM); (f) Detail of teliospores production. Scale bar = 2 µm. "> Figure 4
Multilocus phylogenetic tree of Anthracocystis species inferred from RAxML and Bayesian analysis based on ITS sequences. The bootstraps ≥ 70 and Bayesian posterior probabilities ≥ 0.90 are indicated above the nodes, respectively. Isolate from the study are highlighted in bold. The tree was rooted with Langdonia confusa and Triodiomyces triodiae. The type isolate was identified as “t”, isotype as “i”, and holotype as “h” during isolates identification. "> Figure 5
D. insularis plants 37 days after sowing. Control on the left followed by examples of plants inoculated with Anthracocystis panici-leucophaei on the right. Inoculated plants as follows: teliospores in the soil (TS1), sporidia in the soil (ES3), teliospores on 3–4 leaves plants (TA1), sporidia on 3–4 leaves plants (EA3), teliospores on newly emerged plants (TRE1), and sporidia on newly emerged plants (ERE3). "> Figure 6
Examples of impact of isolates of A. panici-leucophaei on sourgrass growth 90 days after sowing 80 days after inoculation with sporidial suspension on one pair of leaves-plants. (A) Sourgrass plants inoculated with isolate BSV2; (B) Sourgrass plants inoculated with isolate 46. "> Figure 7
Host-specificity evaluation of A. panici-leucophaei. Treated plants appearance at 90 days if age, 60 days after inoculation. (A) Non-inoculated (control) wheat; (B) wheat plants inoculated with the smut fungus; (C) sorghum control; (D) sorghum inoculated with the smut fungus; (E) rice control; and (F) rice inoculated with A. panici-leucophaei. ">

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Abstract

Anthracocystis panici-leucophaei, causal agent of smut on Digitaria insularis (sourgrass), was evaluated as a biological control agent for this weed. Two types of inocula (teliospore and sporidia) were tested to assess its infectivity. The effects of teliospore and sporidia inoculations at different phenological stages of sourgrass were compared, as well as the potential of sporidia and teliospores in post-emergence sourgrass management. Virulence tests were conducted with the isolates obtained from D. insularis and evaluation of specificity of A. panici-leucophaei. Both teliospores and sporidia of A. panici-leucophaei are infective to D. insularis in three different phenological stages. Newly emerged plants with one pair of leaves are more sensitive to A. panici-leucophaei. Infection by A. panici-leucophaei inhibits the growth of sourgrass, decreasing several physiological parameters of D. insularis plants. The fungus produces systematic infection of sourgrass plants and may induce the formation of sori in a significant proportion of the plant panicles, partly castrating those plants. Among sixteen A. panici-peucophaei isolates tested, isolate 46 was the most virulent and inhibited the growth of sourgrass plants, and thus appears to have good potential as a biological control agent to be deployed against sourgrass. A. panici-leucophaei was demonstrated to be specific to D. insularis.

Keywords:

smut; mycoherbicide; sourgrass; weed

1. Introduction

One of the most serious agricultural problems is the infestation of weed species in economically important crops. This is recognized as one of the main limiting factors behind losses in crop production [1]. Brazil has a great diversity of grasses belonging to Digitaria (Poaceae), some of which are common in agroecosystems [2]. Sourgrass (Digitaria insularis) is one of the worst weeds of annual and perennial crops in South America. It is a perennial grass species with a C4 photosynthetic metabolism [3], and can reach high infestation levels and reproduce either via seeds (up to 40,000 seeds/plant/year) or rhizomes [4,5,6].

Chemical management is being jeopardized by the increasingly widespread resistance of populations of D. insularis to glyphosate [5]. This is due to the vast area that is cultivated with glyphosate-resistant transgenic crop varieties and to repeated applications of the same herbicide for consecutive years [7]. Approximately 90% of the total area of soybean (Glycine max L.) in South America is cultivated with glyphosate-resistant transgenic soybean varieties (RR), and glyphosate is applied at least three times per year [8]. One alternative for management of glyphosate-resistant biotypes is the use of acetyl coenzyme A carboxylase (ACCase) inhibitors as herbicides. However, the repeated use of this group of herbicides has also resulted in reports of resistance to ACCase herbicides in D. insularis in Brazil [4,6].

For decades, chemical control has been the predominant strategy for weed management in most major crops [1]; however, with the increasing herbicide-resistance problems in important weeds, novel forms of management became necessary. Biological control is emerging as a key alternative for pesticides worldwide. Several studies have demonstrated the potential of phytopathogens as weed biocontrol agents [9], especially phytopathogenic fungi [10]. Mycoherbicides are herbicides based on a fungus or fungal metabolites, employed as non-chemical alternatives to reduce the use of synthetic chemical substances in weed control [11]. Some mycoherbicides have been developed, registered, and marketed since the 1980s, such as Collego^® (currently with a new name LockDown™), Colletotrichum gloeosporioides (Penz) Sacc. f.sp. aeschynomene, for the control of Aeschynomene virginica L.; Devine^®, Phytophthora palmivora (Butler) Butler, for the control of Morrenia odorata (Hook. and Arn.) Lindle; Biomal^® (=MaIlet WP), Colletotrichum gloeosporioides f.sp. malvae (Penz.) Penz. and Sacc. in Penz., for Malva pusila Sm. (=Malva rotundifolia L.); and CASST¹, Alternaria cassiae Jurair and Khan, for the control of Cassia obtusifolia L. Other bioherbicides based on bacteria and viruses have also been developed for the management of specific weeds: Camperico™, based on the bacteria Xanthomonas campestris pv. poae Egli and Schmidt, for the control of Poa annua L., and an innovative bioherbicide based on a virus, Tobacco mild green mosaic virus (TMGV). This virus produces a lethal hypersensitivity reaction in Solanum viarum Dunal [12].

Smut-causing fungi have been studied as potential biological control agents for various weed species. Some have shown promising results, including Sporisorium ophiuri for the control of Rottboellia cochinchinensis [13] and Sporisorium cruentum for the control of Sorghum halepense [14]. The smut fungi include a large number of plant pathogens that establish obligate biotrophic relationships with their host. Throughout their whole life inside plant tissue, smut fungi keep plant cells alive and acquire nutrients via biotrophic interfaces. Smut fungi have developed specialized strategies for host colonization, including the evolution of a complex infection. Upon mating of two compatible haploid cells, a parasitic mycelium is generated that can detect plant surface cues, which triggers the formation of appressoria essential for penetration into the host. These fungi secrete a range of effectors precisely adapted to the plant organ and cell type being infected to modulate host immunity and promote successful colonization. Infected plants typically have reduced vegetative growth and inhibited seed production [15]. The aim of the present study was to confirm the identity of the fungus associated to smut symptoms on sourgrass as Anthracocystis panici-leucophaei, and to evaluate it as a potential biological control agent.

2. Materials and Methods

2.1. Survey

Samples of D. insularis showing typical smut symptoms were collected from ruderal and agricultural areas in cities located in the states of Minas Gerais and Goias, Brazil, between April 2021 and January 2022 (Figure 1). Panicles of sourgrass bearing typical sori, as well as other disease symptoms, were brought to the Laboratório de Microbiologia e Fitopatologia (LAMIF) at the Universidade Federal de Uberlândia-UFU/Campus Monte Carmelo.

2.2. Isolation

Teliospores were collected from the panicles of D. insularis showing smut symptoms with a sterile fine pointed needle and were transferred to microtubes containing sterilized distilled water supplemented with streptomycin sulfate (5 mg/L). Three serial dilutions were performed, and 100 μL of each suspension was transferred to plates containing yeast malt-agar (YMA) (3 g/L yeast extract; 3 g/L malt extract; 5 g/L soy peptone; 10 g/L glucose; 15 g/L agar) and incubated at 28 °C in the dark. After 24 h, under a GZ-500 stereomicroscope (Leipzig, Várzea Paulista-SP, Brazil) with zoom 7.5× to 50×, four individual germinated teliospores were transferred to new YMA plates and incubated for an additional 48 h. After that, one single colony for each isolate was transferred onto another YMA plate. Pure yeast-like sporidial cultures, obtained through this process, were incorporated in a temporary culture collection.

Fragments of seven-day-old yeast-like colonies produced in YMA, or fresh teliospores collected directly from sori, were kept in a 10% aqueous glycerol solution in a ultrafreezer (−80 °C).

2.3. Taxonomy

2.3.1. Morphological Studies

For the observation of teliospore morphology, microscopic preparations were made using lactophenol cotton blue and lactoglycerol of teliospores present in the sori of two isolates obtained in the survey and selected for taxonomic identification, namely, BSV1 and BSV2, isolates from Monte Carmelo-MG. Subsequently, they were examined under a light microscope (Olympus, BX53, São Paulo-SP, Brazil) fitted with differential interference contrast illumination and equipped with a digital capture system (Olympus Q-Color 5™, São Paulo-SP, Brazil). Biometric measurements of at least 30 teliospores per sample were performed.

Selected sub-samples of dried material containing sporulating sori were mounted on stubs using double-sided adhesive tape and gold-coated with a Balzer’s FDU 010 sputter coater. A scanning electron microscope (SEM) Carl-Zeiss Model LEO VP 1430 (AG, Jena, Germany) was utilized to further examine the ultra-structure of the fungus.

2.3.2. DNA Extraction, PCR Amplification, and Sequencing of Isolates

The genomic DNA of isolates BSV1, BSV2, and 46 (an additional isolate collected in the second round of surveys) was extracted from pure cultures of the three isolates grown on YMA plates, incubated at 25 °C with a 12 h photoperiod for seven days. Approximately 50–80 mg of each fungal colony was scraped from the colonies and transferred to sterilized 1.5 mL plastic microtubes containing zirconium beads. DNA extraction was performed using the Wizard^® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions.

PCR amplification of the the Internal Transcribed Spacer (ITS) region was conducted with primers ITS4 and ITS5 [16]. The PCR reaction was set up with a total volume of 12.5 μL, comprising 2 µL (20 ng) of DNA, 6.25 µL of Dream Taq Master Mix (Fermentas Company, São Paulo, Brazil), 0.25 µL of DMSO, 1.25 µL of BSA, 2.25 µL of H₂O, and 0.25 µL of each primer. PCR cycling consisted of initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were analyzed on 2% agarose gel stained with GelRed™ (Biotium Inc., Hayward, CA, USA) and visualized under UV light to assess amplification size and purity.

PCR products were purified and sequenced by Macrogen Inc., Seoul, Republic of Korea (http://www.macrogen.com, accessed on 25 June 2022). Electropherograms were evaluated, ambiguous nucleotides were corrected, and consensus sequences (contigs) were generated using SeqAssem ver. 07/2008 software [17].

Phylogenetic analysis of the ITS region of the smut from sourgrass was conducted using the Bayesian inference (BI) and maximum-likelihood methods with the Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway V. 3.3 using MrBayes on XSEDE 3.2.6 for BI and RAxML v.8.2.12 [18]. Phylogenetic trees were visualized using FigTree v1.3.1 [19] and edited using CorelDRAW Graphics Suite 2017. Prior to phylogenetic analyses, the best substitution model for the gene/locus was determined using jModelTest2 ver. 2.1.6 [20], and BI analysis employing a Markov chain Monte Carlo method was performed with all sequences. The GTR + I + G evolution model was used for the ITS region, selected based on the Akaike Information Criterion (AIC) after likelihood score calculation with MrMODELTEST 2.3 [21].

Once likelihood scores were calculated, models were selected according to the Akaike Information Criterion (AIC). The GTR + I + G evolution model was used for the ITS region. Phylogenetic analysis of the ITS region was conducted using the Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway V. 3.3 utilizing MrBayes on XSEDE 3.2.6 [18]. The resulting phylogenetic trees were visualized using FigTree v1.3.1 [19] and edited in CorelDRAW Graphics Suite 2017.

2.4. Evaluation of Anthracocystis panici-leucophaei as a Biological Control Agent for Digitaria insularis

In experiments Section 2.4.3 and 2.4.4, the isolates obtained from the initial survey, BSV1 and BSV2 (mixed isolates in water suspension), were used. For the specificity test (Section 2.4.6), isolate BSV2 was utilized. Following new collections and the addition of new isolates to the collection, the aggressiveness of different isolates of A. panici-leucophaei on D. insularis plants was evaluated (Section 2.4.5).

Test plants were grown from seeds obtained at Cosmo Agrícola Produção e Serviços Rurais LTDA in a substrate consisting of sterilized soil + sand + manure in a 1:1:1 ratio kept in 300 mL plastic pots.

2.4.1. Preparation of Teliospores for Use as Inoculum

Selected sori, examined and found free of mycoparasites or mycophagous insects, were crushed with mortar and pestle and a 60-mesh sieve was used to remove plant debris and produce a clean mass of teliospores. A viability tests of teliospores was conducted prior to use in experiments. Percentage germination was assessed by placing a 50 µL aliquot of a teliospore suspension in sterilized distilled water on either microscope slides or Petri dishes containing YMA. These were placed in an incubator at 25 °C either adjusted to a 12 h light regime or in continuous dark, and observations were made after 4 and 12 h.

2.4.2. Sporidial Production

The sporidia were obtained by transfer of small portions of colonies formed on YMA onto 250 mL flasks containing liquid YM (3 g/L yeast extract, 3 g/L malt extract, 5 g/L soy peptone, 10 g of glucose). These were then placed on an orbital shaker at 28 °C and 150 rpm for 5 days to obtain yeast-like sporidia. Sporidial suspensions of BSV1 and BSV2 were then diluted with sterile distilled water (SDW) to 10⁶ sporidia mL⁻¹ and plated together on YMA for compatibility testing. A compatible reaction was recognized by the appearance of white mycelium (supposedly resulting from fusing of compatible sporidia) on a plate after 5 days of incubation. The isolates BSV1 and BSV2 were considered compatible (sporidia of opposite sexual types) as described in Banuett and Herskowitz [22] and Santiago et al. [23].

Such compatible isolates (BSV1 and BSV2) were separately transferred to liquid YM flasks and left on an orbital shaker at 28 °C and 150 rpm for 5 days in the dark to stimulate the multiplication of the sporidia. The concentration of the resulting cell suspension obtained was adjusted to 1 × 10⁶ sporidia/mL with the help of a haemocytometer. A mixture of compatible suspensions from isolates BSV1 and BSV2 was prepared by mixing 50% of the 1 × 10⁶ sporidia/mL suspension volume of each isolate and immediately utilized in inoculation tests.

2.4.3. Comparative Effect of Teliospores vs. Sporidia Inoculations of BSV1 and BSV2 on Digitaria insularis at Different Phenological Stages

Observations on the effect of each type of A. panici-leucophaei inoculum on three different phenological stages of D. insularis (seeds, one pair of leaves, plants, and 3–4 leaf-plants) was made in order to select the ideal inoculum kind and plant stage for establishing a standard for later experiments. Twelve D. insularis seeds were sown in 0.5 L pots containing the mixture mentioned above. Seven days after germination started, the plant stand on each pot was standardized and five plants were left in each pot with excess plants removed. Sporidia of BSV1 and BSV2 were produced as described in Section 2.4.2. Sporidial suspensions involved a mixture of the two isolates of purported compatible mating types. Plants and seeds were sprayed until runoff with a hand-held sprayer. The treatments consisted of: (a) TS1, application of 10 mL/pot of a teliospore suspension of isolate BSV1 (3.23 × 10⁶ teliospores mL⁻¹ + 0.01% Tween^® 20). The application was performed by drenching the seeds with inoculum suspension on the substrate surface (pre-emergence); (b) TRE1, application of 10 mL/pot of a teliospore suspension of isolate BSV1 (3.23 × 10⁶ teliospores mL⁻¹ + 0.01% Tween^® 20), on plants having one pair of leaves (10 days after sowing (DAS)); (c) TA1, Ibid application of BSV1 teliospore suspension on 3–4 leaf-plants (25 DAS); (d) ES3, direct application of 10 mL/pot of a mixed sporidial suspension (BSV1 + BSV2) (3.17 × 10⁶ sporidia mL⁻¹ + 0.01% Tween^® 20) on the seeds and soil surface (pre-emergence); (e) ERE3, Ibid on one pair of leaves-plants (10 DAS); and (f) EA3, Ibid on 3–4 leaves-plants (25 DAS). The control consisted of pots seeded with sourgrass as described above, but only sprayed with sterile distilled water (SDW) + 0.01% Tween^® 20. The plants were kept in a greenhouse at 26–28 °C and irrigated twice a day. In the first 48 h after inoculum application (or spraying with water), the pots were kept in a humid chamber. Evaluation was conducted 37 days after sowing. Plant height and root length, measured with a ruler, stem diameter, measured with a pachymeter, dry biomass of roots and aerial part, evaluated by drying in an oven with air circulation at 65 °C for 48 h, chlorophyll, with a clorofiLOG (Falker^®, Porto Alegre, RS, Brazil), and number of leaves per plant were assessed. The experiment was conducted in a completely randomized design with five plots and 20 repetitions for each treatment, with each plant considered a repetition. Data were subjected to analysis of variance (ANOVA, p < 0.05), and means were compared by Tukey’s test. Reisolation of the fungus was made from five pieces of stunted inoculated plants (1 cm²) by transfer of these fragments in 70% ethanol for 1 min and sodium hypochlorite (2% active chlorine) for 3 min, followed by triple washing in deionized and autoclaved water, and transferred to petri dishes containing potato dextrose agar medium to confirm the presence of A. panici-leucophaei within diseased tissues. The presence of the pathogen was confirmed by the formation of colonies with sporidia of the fungus after 5 days of incubation at 25 °C.

2.4.4. Effect of Post-Emergence Inoculation of Sporidia and Teliospores of Anthracocystis panici-leucophaei on Digitaria insularis

Thirty seeds of D. insularis were sown in each of 5.0 L pots containing the substrate described above. Smut sporidial suspension was applied using a mini-pressurizer on newly emerged plants (10 DAS) with 2–3 leaves. The treatments consisted of: (a) TRE, application of 20 mL/pot of a teliospore suspension of isolate BSV1 (2.31 × 10⁶ teliospores/mL⁻¹ + 0.01% Tween^® 20); (b) BSV1 + BSV2, application of 20 mL/pot of a sporidia suspension of isolates BSV1 and BSB2 in mixture (2.64 × 10⁶ sporidia/mL⁻¹ + 0.01% Tween^® 20); (c) BSV1, application of 20 mL of a sporidia suspension of isolate BSV1 (2.39 × 10⁶ sporidia/mL⁻¹ + 0.01% Tween^® 20); and (d) BSV2, application of 20 mL of a sporidia suspension of isolate BSV2 (2.51 × 10⁶ sporidia/mL⁻¹ + 0.01% Tween^® 20). The control consisted of pots sprayed with sterile distilled water + 0.01% Tween^® 20. All treatments were kept for 48 h in a dew chamber after inoculation. Subsequently, the plants were maintained in a greenhouse at approximately 25 °C under a 12 h photoperiod.

This experiment was performed after 102 days of sowing, until the emergence of healthy panicles or those with smut symptoms (sori with teliospores). Evaluation was conducted based on the following parameters: plant height, root length, stem diameter, and dry weight of root and aerial part (procedure described above). Additionally, the number of leaves per plant, number of tillers, number of inflorescences per plant, and number of tillers bearing at least one sorus were also recorded.

The experiment was conducted in a completely randomized design with five plots and 25 repetitions for each treatment, considering one plant as a repetition. Data were subjected to analysis of variance (ANOVA, p < 0.05), and means were compared by Tukey’s test.

2.4.5. Comparative Virulence of A. panici-leucophaei Isolates on Digitaria insularis

Sixteen different isolates of A. panici-leucophaei which were obtained during the field surveys were included. Sporidial production was conducted as described in Section 2.4.2 and suspended in sterile distilled water + 0.01% Tween^® 20 at a concentration of 1.0 × 10⁶ sporidia/mL.

Suspension was sprayed onto newly emerged sourgrass seedlings grown in pots as described above (10 DAS). Each of the ten plants in each pot had 2–3 leaves. Each pot was sprayed with 20 mL of the sporidial suspension separately with each isolate until runoff. Controls were only sprayed with SDW + 0.01% Tween^® 20. All pots were kept in a dew chamber for 48 h after inoculation, and subsequently transferred to a greenhouse bench.

Evaluation was performed 90 days after sowing as described in Section 2.4.4.

The experiment was conducted in a completely randomized design with 17 treatments (16 isolates plus control), with five repetitions for each treatment. Each pot with 10 seedlings represented one repetition.

Values obtained for each parameter were analyzed/tested for presuppositions, where residual normality was verified using the Shapiro–Wilk test (p < 0.05), and variance homogeneity was assessed using the Bartlett test (p < 0.05). The data were subjected to analysis of variance (ANOVA), and subsequently, the means were compared using the Scott-Knott test (p < 0.05). Comparison of treatment means relative to the control was conducted using the Dunnett test (p < 0.05).

The statistical program used was R software version 4.0.2. ANOVA and Bartlett’s variance homogeneity test were performed using scripts developed for the ExpDes.pt data package version 1.2 [24], while the Shapiro–Wilk residual normality test was executed using scripts from the Nortest data package version 1.0-4 [25]. The Dunnett test was executed using scripts from the multcomp data package version 1.4-20 [26].

2.4.6. Evaluation of Specificity of A. panici-leucophaei to Sourgrass

Twenty-one grass species were selected for a specificity test (Table 1). Plants were inoculated, as described in Section 2.4.3. A larger volume (20 mL/pot) was utilized, and the isolate used in inoculations was BSV2 (1.0 × 10⁶ sporidia/mL). Newly emerged plants (10 DAS) were targeted. After inoculation, the plants were kept in a humid chamber for 48 h and then transferred to a greenhouse bench. For each plant species, six pots with five individual plants were included, with three pots inoculated with BSV2 and three pots serving as controls, only sprayed with a solution of 0.01% Tween^® 20. Evaluation was conducted 90 days after sowing by recording the incidence or absence of disease symptoms: dwarfing and smut symptoms (sori).

3. Results

3.1. Survey

During a survey of phytopathogenic fungi associated with Digitaria insularis in the Alto Paranaíba region (Minas Gerais-Brazil), diseased plants were observed in the municipality of Monte Carmelo, showing severe dwarfing and smutted panicles (castration). The typical formation of a “whip” or sori at the apical meristem of the plants was often observed with panicles covered by a dark mass of spores. Such symptoms are similar to those observed in smutted sugarcane (attack by Sporisorium scitamineum) and as a result of attack of other members of the Ustilaginaceae on members of the Poaceae (Figure 2).

Sixteen samples of D. insularis bearing typical A. panici-leucophaei smut symptoms were obtained and pure cultures were obtained from each, with one isolate being selected from each sample/origin: 13 from the state of Minas Gerais, and 3 from the state of Goias (Brazil) (Table 2).

3.2. Taxonomy

Anthracocystis panici-leucophaei (Bref.) McTaggart and R.G. Shivas [27], MycoBank MB801662. For a complete list of synonyms, see [28].

Notes: On specimens collected here, sori formed on upper leaves of sterile shoots or floral stems or immature inflorescences. Sori were long, cylindrical, often twisted, tapering towards the apex, 0.2–0.8 × 4–15 cm, covered by white to slightly grayish peridium that ruptured at maturity. Parenchymatous tissues between the vessels were replaced by a mass of dark brown, semi-agglutinated, or granular powdery teliospores, revealed by the rupture of the peridium. Teliospores formed chains while immature, globose to subglobose, ellipsoidal to slightly irregular, sub-polyhedral, 6.0 × 8.0 µm, yellowish-brown; uniformly thick and densely verrucose wall (Figure 3).

Specimens examined: Brazil: state of Minas Gerais; Monte Carmelo, on Digitaria insularis, 16 April 2021, B. S. Vieira, BSV1 and BSV2 isolates.

3.3. Phylogenetic Analysis

Sequences from the ITS region of the BSV1, BSV2, and 46 isolates submitted to BLAST showed high similarity (100%, 99.72% and 100%, respectively) with sequence CP060299 from isolate SPL10 of Anthracocystis panici-leucophaei (Bref.) McTaggart and R.G. Shivas.

The phylogenetic tree obtained from Bayesian inference showed that the BSV1, BSV2, and 46 isolates are grouped with high support (0.98) and 96% of bootstrap with other isolates of A. panici-leucophaei, which in turn formed a well-supported clade (1.0) within the genus Anthracocystis (Figure 4).

3.4. Evaluation of Anthracocystis panici-leucophaei as a Biological Control Agent for Digitaria insularis

3.4.1. Comparative Effect of Teliospores vs. Sporidia Inoculations of BSV1 and BSV2 on D. insularis at Different Phenological Stages

Both types of Anthracocystis panici-leucophaei inocula were infective to D. insularis. The infectivity of the two types of A. panici leucophaei inocula was confirmed by re-isolating the fungus from pieces of stunted inoculated plants. The presence of the pathogen was confirmed by the formation of typical yeast-like sporidial colonies after 5 days of incubation at 25 °C.

Inoculation involving either teliospores or sporidia in pre-emergence, on newly emerged plants, and on the aerial part (plants with 3–4 leaves) led to a delay in the development of the plants. The height of the sourgrass plants was reduced in all treatments as compared to the control (Table 3).

The treatment with sporidia in newly emerged plants resulted in the lowest plant height, with 65% inhibition as compared to the control. Root length was similarly affected in all treatments where the pathogen was inoculated in all phenological stages. Root length in treatment ERE3 was 40.9% lower compared to the control. As for stem diameter, except for the treatment with teliospores inoculated in the soil, all other treatments exhibited smaller stem diameters, indicating that the fungus was able to infect the sourgrass plants in all three phenological stages tested, resulting in less vigorous plants.

Similarly, the dry root mass weight was reduced in all treatments as compared to the control. The post-emergence treatments (25 DAS) EA3 and TA1 resulted in reductions in root mass of 86.9% and 86.4%, respectively, as compared to the control, representing the lowest dry root mass weights. Although the other treatments did not differ significantly from each other, the treatments on newly emerged plants, ERE3 and TRE1, showed lower inhibition percentages, with reductions of 80.9% and 78.7%, respectively, with higher dry root mass weights. The dry aerial part mass (DAPM) showed the highest variability among treatments. Inoculation of sporidia and teliospores on newly emerged plants (ERE3 and TRE1) resulted in reductions of 80.6% and 73.5%, respectively, as compared to the control. This was not significantly different from treatments EA3 and TA1, inoculated at post-emergence (25 DAS), which showed reductions of 64.1% and 63.7%, respectively. TS1was the treatment that produced the smaller reduction in dry weight of the aerial part of sourgrass (47.5%).

Significant changes were observed in the chlorophyll A index in sourgrass plants treated with sporidia and teliospores in the three phenological stages included in the test. The lowest chlorophyll A index was observed in the pre-emergence treatment TS1, showing a reduction of 73.6% compared to the control. The other treatments (ES3, TRE1, ERE3, TA1, and EA3) did not exhibit significant differences from each other regarding this variable; however, the chlorophyll A index decreased by 54.4%, 53.8%, 56.7%, 56.9%, and 48.6% as compared to the control, respectively. The number of leaves per plant was higher in plants where teliospores were inoculated into the soil (5.35 leaves/plant), not differing from ES3 and TA1 and being higher than the control (Table 3). The treatment with the lowest number of leaves was TRE1 (3.95 leaves/plant).

Simple visual observation of the plants involved in the study showed evident inhibition in the development of the aerial part of D. insularis plants in treatments TRE1, ERE3, and EA3 (Figure 5), demonstrating that the two fungal structures were capable of infecting plants at different phenological development stages. The infectivity of the two types of A. panici-leucophaei inocula was confirmed by re-isolating the fungus from pieces of stunted inoculated plants.

3.4.2. Effect of Post-Emergence Inoculation of Sporidia and Teliospores of Anthracocystis panici-leucophaei on Digitaria insularis

One hundred and two days after sowing (DAS), the panicles had appeared on plants in all treatments. It then was found that the spraying of sporidia with the isolate BSV2 produced the most significant reduction in the height of sourrass plants, with an average 40.3% reduction as compared to the control. This was also the only treatment that led to sori formation on inflorescences (Table 4). The other treatments (TRE, BSV1, and BSV1 + BSV2) did not exhibit statistical differences from the control, and did not lead to sori being produced on plants; however, some reduction in plant height was also observed for these treatments.

These results suggest that the isolate BSV2 has better potential for biocontrol of D. insularis than BS1, and that their combined use is inadequate.

Either teliospores or sporidia of BSV2 or sporidia of BSV1 reduced the root length of D. insularis. with inhibitions of 21.5%, 21.9%, and 23.6% as compared to the control in the TRE, BSV1, and BSV2 treatments, respectively. Only the combined use of BSV1 and BSV2 did not produce a reduction in root length, being significantly similar to the control (Table 4).

Plants in the BSV2 and TRE treatments exhibited the greatest reductions in stem diameter, with 15.8% and 14.7%, respectively. The BSV1 and BSV1 + BSV2 treatments reduced stem diameter by 14.0% and 10.2%, respectively.

The number of tillers in the BSV2 treatment was higher than all others, including the control. For the TRE, BSV1, and BSV1 + BSV2 treatments, the number of tillers was lowerered by 21.3%, 9.3%, and 8.0%, respectively, as compared to the average number of tillers in the control. All treatments exhibited a significantly lower number of tillers bearing inflorescence as compared with the control. The treatment involving teliospore applications led to a reduction in the number of fertile tillers (78.6% reduction), followed by the BSV2 (64.3% reduction), BSV1 (35.7% reduction), and BSV1 + BSV2 (35.7% reduction) treatments. It was observed that only plants treated with BSV2 had panicles bearing sori. For those, approximately 68% of the plants had typical active A. panici-leucophaei sori. Such results are interpreted here as an excellent indication of the biocontrol potential of BSV2 against sourgrass.

BSV2 treatments reduced the dry root mass of sourgrass plants by 65.8% as compared to the control. On the other hand, TRE, BSV1, and BSV1 + BSV2 treatments reduced root dry biomass, respectively, by 41.9%, 31.5%, and 34.5%. Similarly, a significant decrease in the dry aerial part mass of sourgrass plants was observed for all treatments. For BSV2, TRE, BSV1 + BSV2, and BSV1, the reductions in dry aerial biomass of sourgrass were, respectively, 51.3%, 44.4%, 43.0%, and 37.2%. BSV2 application produced the greatest reductions in root and aerial dry biomass and also in number of leaves per plant (reduction of 30.6%) as compared to the control.

3.4.3. Comparative Virulence of A. panici-leucophaei Isolates on Digitaria insularis

Ninety days after inoculation, all 16 isolates of A. panici-leucophaei included in the study had produced sori on at least one of the plants included in the test. Whip-like sori replaced the inflorescences of infected plants. Panicles became covered by a powdery mass of dark teliospores, confirming that all 16 isolates are capable of producing systemic infections on D. insularis triggered by sporidial inoculations.

The comparison performed here indicated that isolate 46 was the most virulent under the conditions under which the test was performed. It reduced plant height by 45% as compared to the control (Table 5). Isolates 31, BSV2, and 37-1 also produced significative reductions in plant growth, reducing their average height by 23%, 42%, and 24%, respectively.

Treatment with isolate 46 reduced the length the roots of D. insularis by 61% as compared to the control. Conversely, treatment with isolates 11-1, 16-1, 3-1, 36-1, 65, 64, 47, and BSV2 did not produce significant reductions in root development.

Application of isolates 46, 37-1, 31, and 49 on sourgrass led to the greatest reductions in stem diameter: 47%, 38%, 51%, and 50%, respectively.

The number of leaves of sourgrass treated with the smut fungus was significantly reduced by applications of isolates 11-1 (27.13% reduction) and 46 (24.49% reduction) as compared with the control.

The number of tillers in plants treated with 15-1 was significantly higher than for all other isolates, including the control.

Dry aerial part mass (DAPM) was equivalent to control for two isolates (BSV1 and 64), whereas applications of all other isolates led to a reduction in the aerial biomass of sougrass at levels ranging from 81% for 46 to 51.42% for 36-1 (Table 6). Dry root biomass was also reduced by application of A. panici-leucophaei isolates. Reductions ranged from 57% for 31 to 39% for isolate 46. Isolate 46 demonstrated the highest reduction in the dry aerial part compared to the control, and treatment with isolate 31 showed the highest reduction in dry root mass.

Isolates 49, 64, and 57 formed the largest number of sori per plant. That ability is of interest as it represents a “partial castration” of infected individuals, which may lead to reduction in the quantity of sourgrass seeds produced in a field. On the other hand, plants treated with isolates BSV2, 31, 37-1, 46, 11-1, 36-1, 65, 49, and 46 produced fewer inflorescences per plant than non-inoculated plants. Reductions ranged from 64.70% for BSV2 to 14.51% for isolate 46.

Based on all evaluations performed in this work, it appears that isolate 46 is the most virulent among the 16 isolates under evaluation, considering the combination of all features that were evaluated and the conditions under which the experiment was conducted (Figure 6).

3.4.4. Evaluation of Specificity of A. panici-leucophaei to Sourgrass

None of the 21 species of the Poaceae family developed disease symptoms after inoculation with one representative isolate of A. panici-leucophaei (BSV2) (Figure 7). Thus, A. panici-leucophaei has shown to be host-specific and limited to infection and completion of its cycle on D. insularis, which is a relevant characteristic in a biocontrol agent, as it demonstrates its ability to control only the target species without affecting other crops.

4. Discussion

The adaptation of sourgrass to various environmental conditions and the increasing occurrence of weed populations that are resistant to glyphosate and ACCase-inhibitor herbicides in different agricultural areas in Brazil is a major cause of concern for farmers [5]. As this plant has rapid seed and rhizome production, it is recommended that its management should be carried out within 35 days after emergence [29,30].

Anthracocystis panici-leucophaei (=Ustilago panici-leucophaei) was described in Digitaria insularis (=Panicum leucophaeum), in Rio de Janeiro, Brazil, by Brefeld in 1895 [31]. Recently, RWB briefly attempted to recollect material from Rio de Janeiro, but without success. The continuation of the search for material from Rio de Janeiro is justifiable as a neotype still needs to be designated, since Brefeld’s type material no longer exists in the Hamburg herbarium (HBG).

The morphological features of isolates BSV1 and BSV2, chosen as examples of the population of the smut fungus on sourgrass in the state of Minas Gerais, were very close to those described for Anthracocystis panici-leucophaei by Vánky [28], Cunnington et al. [32], and Piatek [33]. Anthracocystis panici-leucophaei was reported as a pathogen of Digitaria ammophila, D. breviglumis, and D. brownii in Australia, of D. insularis in Cuba and Brazil, Echinochloa polystachya in Argentina, Oplismenopsis najada in Paraguay, Paspalum saccharoides in Colombia, Triachne californica in Cuba and the United States, Triachne insularis in Cuba, the Dominican Republic, and the United States, and Trichachne insularis in Brazil and Colombia [34].

The genus Anthracocystis has undergone numerous changes over the years. Many species previously belonging to other genera have been included in the group, as a result of imprecise or developing taxonomy for smut-causing fungi. Many revisions have been made in an attempt to clarify the phylogenetic position of these fungi [27,32,35,36]. Interestingly, McTaggart et al. [27], when promoting the recombination of Sporisorium panici-leucophaei to A. panici-leucophaei, did not include isolates from the type locality in their phylogenetic analysis. Despite this, our isolates collected in Brazil grouped with the others, corroborating the results that S. panici-leucophaei indeed fits well in Anthracocystis.

Information about the life cycle of this fungus is lacking. Therefore, issues of great practical relevance for its use as a biocontrol agent have remained unknown. Issues such as the requirement of different mating types and heterokaryosis before infection and the susceptibility of host stages, among others, are only now being elucidated. Its resemblance to Sporisorium scitamineum would suggest an equivalent biology. The sugarcane smut fungus presents three different morphological phases, namely, yeast-like haploid sporidia, dicaryotic hyphae, and diploid teliospores [37]. Under optimal environmental conditions, teliospores germinate, undergo meiosis, and produce haploid sporidia that grow in culture medium like yeast cells. According to Alexander and Srinivasan [38], infection in the host occurs when there is a combination of two haploid sporidia of opposite mating types, forming infective dicaryotic hyphae. In the present work, both inoculations with the mixture of isolates BSV1 and BSV2 and inoculations with isolates separately resulted in systemic infection in D. insularis plants, resulting in plants forming sori with teliospores. It appears that A. panici-leucophaei is homothallic or has a form of mating-type system that allows single sporidial colonies to remain infective. This renders its manipulation and use simpler under the biocontrol perspective.

The potential for infection of teliospores and sporidia of smut-causing fungi studied for use in biological control has been studied for various target-weeds worldwide. Some notable examples are Sporisorium ophiuri for the control of Rottboellia cochinchinensis [13]; Sphacelotheca holci for the control of Sorghum halepense [39]; and Sporisorium cruentum for the control of Sorghum halepense [14,40].

Millhollon [14] evaluated the effect of inoculating teliospores of Sporisorium cruentum injected into Sorghum halepense seedlings from 14 to 21 days after sowing (DAS), in contrast with teliospore spraying on plants 45 to 90 cm tall, at the initial flowering stage, along with the interference of the weed on the yield of Saccharum sp. hybrids. S. halepense seedlings injected with S. cruentum teliospores showed 98% disease incidence in the first year of cropping, and cane production was of 57,000 kg ha⁻¹, whereas cane production in areas infested with S. halepense but not treated with the smut was of 37,000 kg ha⁻¹. The average of infected S. halepense plants in areas sprayed with teliospore suspension was of only 54%, with a smaller gain in cane production (yield of approximately 52,000 kg ha⁻¹).

Systemic infection of A. panici-leucophaei in sourgrass was shown here to drastically inhibit the physiological growth parameters of this weed, particularly when the pathogen was inoculated in newly emerged plants. This potentially provides a competitive advantage for plantations in field conditions. In a real-field condition, spraying sougrass with A. panici-leucophaei sporidia in post-emergence between crop rows with the aim of reducing the vigor of sourgrass individuals may be sufficient to contain the damage caused by this troublesome weed.

Additionally, A. panici-leucophaei infection considerably reduces the formation of panicles in sourgrass plants and may substitute the reproductive structures of infected plants with sori, partly castrating such plants. It is evident that A. panici-leucophaei infection reduces seed production in previously inoculated sourgrass plants. Digitaria insularis has a high seed production covered in dense pilosity with high germination power which can be dispersed over long distances by wind, practically year-round. Sourgrass is very prolific, as under normal conditions it produces up to 40,000 seeds/plant/year [6]. Additionally, these seeds can germinate over a wide range of temperatures and light intensities, significantly increasing species dissemination [41]. The reduction in seed production is highly desirable for any method of weed control, particularly in the case of D. insularis. It can be conjectured that repeated applications of A. panici-leucophaei in an infested area will progressively decrease the sougrass seed bank in the medium to long run.

A significant reduction in the aerial part of sourgrass plants was obtained after the inoculation of A. panici-leucophaei sporidia in newly emerged plants, including percentages of aerial plant inhibition greater than that obtained with applications of chemical herbicides [42]. For example, inoculation of sporidia in newly emerged sourgrass plants resulted in an over 80% reduction in dry aerial part mass compared to non-inoculated plants. Coradin et al. [42] evaluated the dry mass of aerial part sourgrass plants subjected to pre-emergence herbicide application and found that the herbicide diclosulam at the recommended dose reduced the dry aerial part mass of plants by 58.3% compared to the control in plants evaluated at 28 days after emergence (DAE). We acknowledge that the comparison between the fungus and the chemical herbicide is tenuous. Nevertheless, it indicates that A. panici-leucophaei may become a viable active ingredient for a mycoherbicide in the future.

In the present study, root length in the ERE3 treatment (direct application of a mixed sporidial suspension (BSV1 + BSV2) on one pair of leaves-plants (newly emerged sourgrass plants)) was over 40.0% less than in the control, indicating that sporidia were infective to the aerial part of sourgrass plants and that the inoculation targeting the aboveground plant parts have an indirect impact on below ground plant parts, inhibiting root development.

Massion and Lindow [39] showed that one of the effects of Sphacelotheca holci infection on the morphology of the weed plant Sorghum halepense was a reduction in aerial part and rhizome length by 28.8% and 44.5%, respectively. Gassó et al. [40] showed that there was a 50% reduction in the dry mass of S. halepense rhizomes from plants inoculated with S. cruentum teliospores, while for sporidia the reduction was 43.6% for rhizome dry mass weight.

Johnson and Baudoin [43] conducted greenhouse experiments to evaluate the potential of the fungus Ustilago syntherismae as a biological control agent for Digitaria ciliaris. The incidence of infected plants, sown 1 cm deep and between 0 to 22 days before application of U. syntherismae teliospores, was evaluated. The highest percentage of infected plants occurred when inoculation was performed on the same day as sowing, at three inoculum concentrations (3.6 × 10⁷, 7.0 × 10⁶, and 1.8 × 10⁶ teliospores mL⁻¹), with incidence rates of approximately 86%, 81.5%, and 57%, respectively. When D. ciliaris sowing was conducted between 7 to 22 DAA, there was a reduction in the number of infected plants. This reveals that the phenological stage of the plants can interfere with the fungus’s infection capacity in the target plant.

Massion and Lindow [39] evaluated the effect of Sphacelotheca holci infection on the morphology and competitiveness of Sorghum halepense. They found that smut infection reduced the aerial dry biomass as well as root, and rhizome length by 28.8%, 49.0%, and 44.5%, respectively, compared to non-inoculated plants. Non-infected plants were noticeably more vigorous. When in direct competition with corn smut-infected targets, weeds had their development diminished dramatically. Aerial dry biomass suffered a reduction of 68.3%, root length was 76.5% lower than the control, and rhizome length was reduced by 81.9%.

Millhollon [14] evaluated the potential of teliospores and sporidia of Sporisorium cruentum for the biocontrol of Sorghum halepense, demonstrating infection superior to 90% of plants over a period of three years in pot experiments and with greater variability in the field experiment, in the periods of 1997 and 1998, 40% and 88%, respectively, for teliospores and 62% and 71%, respectively, for sporidia.

Gassó et al. [40] evaluated the effect of inoculating a suspension of teliospores and sporidia of the fungus Sporisorium cruentum in Sorghum halepense rhizomes, and found a reduction in the dry matter of the aerial part of the plants by 69.2% and 67.3%, respectively. The same authors observed a reduction in the height of Sorghum halepense plants infected by Sporisorium cruentum compared to the control, which subsequently resulted in a lower aboveground dry matter weight. It is surprising that such results did not lead to the development of a commercial product, considering that the target-weed, Sorghum halepense, is often listed as one of the world’s worst weeds.

Field tests such as those described by Massion and Lindow [39] are still pending for the pathosystem under investigation here, but it is expected that the performance of A. panici-leucophaei as a biocontrol of sourgrass will be significantly improved in a crop-competition situation.

Management methods that decrease the initial development of weeds contribute to postponing future post-emergence herbicide applications, as well as promoting more favorable conditions for economically important crops such as soybeans and corn to close the canopy which can reduce losses in productivity due to weed competition. The discovery of potential control agents and the development of herbicides based on natural products are socially and environmentally desirable [44]. Mycoherbicides have been on hold for several decades; however, now, stimulated by the increasing agronomic interest in alternatives that may allow the control of weeds resistant to chemical herbicides while reducing the environmental impact of such pesticides, the tide may be changing [45].

It can be inferred that isolates of A. panici-leucophaei have high inhibition potential, and the infection of this fungus in newly emerged seedlings interferes negatively with the growth of sourgrass plants.

Timing of inoculation may be critical for the establishment of smut infections. Publications which focus on smuts as crop problems, such as Fischer and Holton [46], have emphasized the importance of inoculation of crop plants during experiments on smut diseases being performed in the early phenological stages of the plants (particularly cereals), since this is commonly the period when they are susceptible to infection by smut fungi. Our smut fungus A. panici-leucophaei is seemingly easier to manipulate. Both teliospores and sporidia can act as infective units, mainly in newly emerged sourgrass plants. In another pathosystem, Johnson and Baudoin [43] observed a high incidence of smut, caused by the fungus Ustilago syntherismae, on Digitaria ciliaris only when the period between the sowing of the weed and inoculation did not exceed four days and the planting depth was not more than 1.0 cm. Further studies are still needed to better clarify the limitations of A. panici-leucophaei as a biocontrol agent against sourgrass; however, under our controlled conditions, such limitations are yet to appear.

Another physiological parameter evaluated in the present work was the number of tillers in sourgrass plants. For example, the number of tillers in the treatment with the BSV2 was higher than in all other treatments, including in the control. The production of multiple tillers, 32 days after inoculation, may characterize an abnormality caused by the infection of A. panici-leucophaei, which subsequently resulted in small panicles with sori. Fungi causing smut symptoms can induce, in addition to the presence of sori, dwarfing of plants, excessive tillering, and, more rarely, plant death [47]. Gassó et al. [40] showed that Sorghum halepense plants in the reproductive phase, 18 to 25 days after inoculation with Sporisorium cruentum, produced multiple tillers (between three to seven tillers) originating from a common node, which gave rise to small infected or sterile panicles, a typical abnormality of S. cruentum infection. Such additional tillers are not in itself a negative result for future management of sourgrass in the field, particularly if the overall size of the plants is reduced while the plant spends its reserves producing supplementary tillers. We can envisage such plants as being less competitive with the invaded crop reducing the losses caused by the weed.

Studies leading to the development of mycoherbicides commonly include, as one of the stages, the selection of fungal isolates with high aggressiveness [48]. The selected isolate should lead to high disease severity in most, if not all, of the target weed plants. Thus, the search for isolates with a higher virulence level is very important. High virulence is a fundamental characteristic of an effective biological control agent [49,50]. Isolate 46 of A. panici-leucophaei was the most aggressive among the isolates tested. New trials should be conducted in field conditions and in micro-plots involving a formulated mycoherbicide prototype containing sporidia of A. panici-leucophaei (isolate 46) to verify whether the promising results on D. insularis, obtained under controlled conditions, can be confirmed.

Smut-causing fungi are generally regarded as examples of pathogens having high levels of host specificity, similarly to the Pucciniales (rust fungi) [47]. In addition to the performance of A. panici-leucophaei in the field, it is essential to further evaluate its specificity. Johnson and Baudoin [43] emphasized that before a pathogen is used as a biological control agent, the range of hosts must be investigated in detail to determine the risk to non-target plants. None of the 21 species of the Poaceae included in our study became diseased after being inoculated with A. panici-leucophaei. Only sourgrass became infected after inoculated with A. panici-leucophaei. Millhollon [14], in his studies on the host range of Sporisorium cruentum, inoculated teliospores into various Sorghum bicolor genotypes to check if they were susceptible to the smut fungus under evaluation for the biocontrol of S. halepense. Seventeen sorghum genotypes tested were not susceptible, and two genotypes were susceptible. The closeness of S. halepense to cultivated species of sorghum complicated this particular study, a matter of less relevance for the case of D. insularis. In the case of our study, there is little room for conflicts of interest, even if it is proven that A. panici-leucophaei infects other species of Digitaria. The fungus is a native from Brazil and occurs freely in nature, and there are no issues related to its introduction as an exotic biocontrol agent. Impact on some other weedy species of Digitaria spp., in case it occurs, would be a bonus for weed management. There is a need to further expand the study involving mainly other grasses and also conducting indirect isolations to verify any internal colonization of tissues from other plants, even if without damage to the host plant.

5. Conclusions

The experiments conducted so far have demonstrated that A. panici-leucophaei has potential as a biological control agent for D. insularis. Definitions of commercial doses, spray volume, adjuvants, prototype formulation, fungus performance under field conditions, compatibility with chemical herbicides, mass production of the fungus, and shelf-life tests should be conducted to confirm the real potential of A. panici-leucophaei as a biocontrol agent for sourgrass.

Author Contributions

Conceptualization, A.P.d.S., J.F.A., E.M.I., B.W.F. and T.F.d.N.; methodology, A.P.d.S., J.F.A., E.M.I. and B.W.F.; validation, A.P.d.S., J.F.A. and F.G.; investigation, A.P.d.S., J.F.A., E.M.I., F.G., B.W.F. and T.F.d.N.; data curation, A.P.d.S., J.F.A. and E.M.I.; writing—original draft preparation, A.P.d.S., J.F.A., E.M.I., B.W.F., T.F.d.N., C.C.M. and B.S.V.; writing—review and editing, A.P.d.S., J.F.A., E.M.I., F.G., B.W.F., T.F.d.N., C.C.M., R.W.B. and B.S.V.; supervision, R.W.B. and B.S.V.; project administration, B.S.V.; funding acquisition, R.W.B. and B.S.V. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Koppert do Brasil Holding LTDA and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

Author Camila Costa Moreira was employed by the company Koppert Do Brasil Holding Ltda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Cimmino, A.; Masi, M.; Evidente, M.; Superchi, S.; Evidente, A. Fungal phytotoxins with potential herbicidal activity: Chemical and biological characterization. Nat. Prod. Rep. 2015, 32, 1629–1653. [Google Scholar] [CrossRef] [PubMed]
Mondo, V.H.V.; Carvalho, S.J.P.; Dias, A.C.R.; Filho, J.M. Efeitos da luz e temperatura na germinação de sementes de quatro espécies de plantas daninhas do gênero. Digitaria. Rev. Bras. Sementes 2010, 32, 131–137. [Google Scholar] [CrossRef]
Kissmann, K.G.; Groth, D. Plantas Infestantes e Nocivas, 2nd ed.; BASF: São Paulo, Brazil, 1997. [Google Scholar]
Gemelli, A.; Junior, R.S.O.; Constantin, J.; Braz, G.B.P.; Jumes, T.M.C.; Neto, A.M.O.; Dan, H.A.; Biffe, D.F. Aspectos da biologia de Digitaria insularis resistente ao glyphosate e implicações para o seu controle. Rev. Bras. Herb. 2012, 11, 231–240. [Google Scholar] [CrossRef]
Lopes-Ovejero, R.F.L.; Takano, H.K.; Nicolai, M.; Ferreira, A.; Melo, M.S.C.; Cavenaghi, A.L.; Christoffoleti, P.J.; Oliveira, R.S. Frequency and dispersal of glyphosate-resistant sourgrass (Digitaria insularis) populations across Brazilian agricultural production areas. Weed Sci. 2017, 65, 285–294. [Google Scholar] [CrossRef]
Takano, H.K.; Melo, M.S.C.; Ovejero, R.F.L.; Westra, P.H.; Gaines, T.A.; Dayan, F.E. Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors. Pestic. Biochem. Physiol. 2020, 164, 1–6. [Google Scholar] [CrossRef] [PubMed]
Timossi, P.C. Manejo de rebrotes de Digitaria insularis no plantio direto de milho. Planta Daninha 2009, 27, 175–179. [Google Scholar] [CrossRef]
Peterson, M.A.; Collavo, A.; Ovejero, R.; Shivrain, V.; Walsh, M.J. The challenge of herbicide resistance around the world: A current summary. Pest Manag. Sci. 2018, 74, 2246–2259. [Google Scholar] [CrossRef] [PubMed]
Charudattan, R. Biological control of weeds by means of plant pathogens: Significance for integrated weed management in modern agro-ecology. BioControl 2001, 46, 229–260. [Google Scholar] [CrossRef]
Klaic, R.; Kuhn, R.C.; Foletto, E.L.; Prá, V.D.; Jacques, R.J.S.; Guedes, J.V.C.; Treichel, H.; Mossi, A.J.; Oliveira, D.; Oliveira, J.V.; et al. An overview regarding bioherbicide and their production methods by fermentation. In Fungal Biomolecules, 1st ed.; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2015; pp. 183–199. [Google Scholar] [CrossRef]
Saxena, S.; Kumar, M. Evaluation of Alternaria alternata ITCC4896 for use as mycoherbicide to control Parthenium hysterophorus. Arch. Phytopathol. Plant Protect. 2010, 43, 1160–1164. [Google Scholar] [CrossRef]
Kremer, R.J. Bioherbicide development and commercialization: Challenges and benefits. In Development and Commercialization of Biopesticides; Academic Press: Cambridge, MA, USA, 2023; Volume 1, pp. 119–148. [Google Scholar] [CrossRef]
Smith, M.C.; Reeder, R.H.; Thomas, M.B. A model to determine the potential for biological control of Rottboellia cochinchinensis with the head smut Sporisorium ophiuri. J. Appl. Ecol. 1997, 34, 388–398. [Google Scholar] [CrossRef]
Millhollon, R. Loose kernel smut for biocontrol of Sorghum halepense in Saccharum sp. hybrids. Weed Sci. 2000, 48, 645–652. [Google Scholar] [CrossRef]
Xia, W.; Yu, X.; Ye, Z. Smut fungal strategies for the successful infection. Microb. Pathog. 2020, 142, 104039. [Google Scholar] [CrossRef]
White, T.J.; Bruns, T.D.; Lee, S.B.; Taylor, J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Academic Press: Cambridge, MA, USA, 1990; Volume 18, pp. 315–322. [Google Scholar] [CrossRef]
Hepperle, D. SeqAssem©. Win32–Version. A Sequence Analysis Tool Contig Assembler and Trace Data Visualization Tool for Molecular Sequences. 2004. Available online: http://www.sequentix.de (accessed on 23 February 2023).
Miller, M.A.; Pfeiffer, W.; Schwartz, T. The CIPRES science gateway: A community resource for phylogenetic analyses. In Proceedings of the 2011 TeraGrid Conference: Extreme Digital Discovery, Salt Lake City, UT, USA, 18 July 2011; pp. 1–8. [Google Scholar] [CrossRef]
Rambaut, A. FigTree, version 1.2.2; Institute of Evolutionary Biology, University of Edinburgh: Edinburgh, UK, 2009. Available online: http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 23 February 2023).
Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef] [PubMed]
Posada, D.; Buckley, T.R. Model selection and model averaging in phylogenetics: Advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst. Biol. 2004, 53, 793–808. [Google Scholar] [CrossRef] [PubMed]
Banuett, F.; Herskowitz, I. Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc. Natl. Acad. Sci. USA 1989, 86, 5878–5882. [Google Scholar] [CrossRef] [PubMed]
Santiago, R.; Alarcón, B.; Armas, R.; Vicente, C.; Legaz, M.E. Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia. Physiol. Plant. 2012, 145, 245–259. [Google Scholar] [CrossRef]
Ferreira, E.B.; Cavalcanti, P.P.; Nogueira, D.A. Package ‘ExpDes.pt’. 2010. Available online: https://cran.r-project.org/web/packages/ExpDes.pt/index.html (accessed on 23 February 2023).
Gross, J.; Ligges, U. Package ‘nortest_1.0-4.tar.gz’. 2015. Available online: https://cran.r-project.org/web/packages/nortest/index.html (accessed on 23 February 2023).
Hothorn, T. Package ‘multcomp_1.4-19.tar.gz’. 2022. Available online: https://cran.r-project.org/src/contrib/Archive/multcomp/ (accessed on 23 February 2023).
Mctaggart, A.R.; Shivas, R.G.; Geering, A.D.W.; Callaghan, B.; Vánky, K.; Scharaschkin, T. Soral synapomorphies are significant for the systematics of the Ustilago-Sporisorium-Macalpinomyces complex (Ustilaginaceae). Pers. Mol. Phylogeny Evol. Fungi 2012, 29, 63–77. [Google Scholar] [CrossRef] [PubMed]
Vánky, K. New smut fungi (Ustilaginomycetes) from Mexico, and the genus Lundquistia. Fungal Divers. 2004, 17, 159–190. [Google Scholar]
Correia, N.M.; Durigan, J.C. Manejo químico de plantas adultas de Digitaria insularis com glyfosate isolado e em misturas com chlorimuronethyl ou quizalofop-p tefuril em área de plantio direto. Bragantia 2009, 68, 689–697. [Google Scholar] [CrossRef]
Machado, A.F.L.; Ferreira, L.R.; Ferreira, F.A.; Fialho, C.M.T.; Santos, L.D.T.; Machado, M.S. Análise de crescimento de Digitaria insularis. Planta Daninha 2006, 24, 641–647. [Google Scholar] [CrossRef]
Brefeld, O. Hemibasidii. Brandpilze III. Cultur der Brandpilzen in Nährlösungen. Untersuchungen Aus Dem Gesammtgebiete Der Mykol. 1895, 12, 99–236. [Google Scholar]
Cunnington, J.H.; Vánky, K.; Shivas, R.G. Lundquistia is a synonym of Sporisorium (Ustilaginomycetes). Mycol. Balc. 2005, 2, 95–100. [Google Scholar]
Piątek, M. Sporisorium kenyanum, a new smut fungus with long twisted sori on Setaria pallide-fusca in Kenya. Pol. Bot. J. 2006, 51, 159–164. [Google Scholar]
Farr, D.F.; Rossman, A.Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Available online: https://nt.ars-grin.gov/fungaldatabases/ (accessed on 4 January 2022).
Piątek, M.; Lutz, M.; Yorou, N.S. A molecular phylogenetic framework for Anthracocystis (Ustilaginales), including five new combinations (inter alia for the asexual Pseudozyma flocculosa), and description of Anthracocystis grodzinskae sp. nov. Mycol. Prog. 2015, 14, 88. [Google Scholar] [CrossRef]
Stoll, M.; Piepenbring, M.; Begerow, D.; Oberwinkler, F. Molecular phylogeny of Ustilago and Sporisorium species (Basidiomycota, Ustilaginales) based on internal transcribed spacer (ITS) sequences. Can. J. Bot. 2003, 81, 976–984. [Google Scholar] [CrossRef]
Benevenuto, J.; Longatto, D.P.; Reis, G.V.; Mielnichuk, N.; Palhares, A.C.; Carvalho, G.; Saito, S.; Quecine, M.C.; Sanguino, A.; Vieira, M.L.C.; et al. Molecular variability and genetic relationship among Brazilian strains of the sugarcane smut fungus. FEMS Microbiol. Lett. 2016, 363, fnw277. [Google Scholar] [CrossRef] [PubMed]
Alexander, K.; Srinivasan, K. Sexuality in Ustilago scitaminea Syd. Curr. Sci. 1966, 35, 603–604. [Google Scholar]
Massion, C.L.; Lindow, S.E. Effects of Sphacelotheca holci infection on morphology and competitiveness of johnsongrass (Sorghum halepense). Weed Sci. 1986, 34, 883–888. [Google Scholar] [CrossRef]
Gassó, M.M.A.; Lovisolo, M.; Perelló, A. Effects of loose kernel smut caused by Sporisorium cruentum onrhizomes of Sorghum halepense. J. Plant Prot. Res. 2017, 57, 62–71. [Google Scholar] [CrossRef]
Mendonça, G.S.; Martins, C.C.; Martins, D.; Costa, N.V. Ecophysiology of seed germination in Digitaria insularis ((L.) Fedde). Rev. Ciência Agron. 2014, 45, 823–832. [Google Scholar] [CrossRef]
Coradin, J.; Braz, G.B.P.; Machado, F.G.; Silva, A.G.; Sousa, J.V.A. Herbicidas aplicados em pré-emergência para o Controle de milho voluntário e capim-amargoso. Rev. Cient. Rural 2019, 21, 51–64. [Google Scholar] [CrossRef]
Johnson, D.A.; Baudoin, A.B. Mode of infection and factors affecting disease incidence of loose smut of crabgrass. Biol. Control 1997, 10, 92–97. [Google Scholar] [CrossRef]
Gomes, F.M.; Fortes, A.M.T.; Silva, J.; Bonamigo, T.; Pinto, T.T. Efeito Alelopático da Fitomassa de Lipinus angistifolius (L.) sobre a germinação e o desenvolvimento inicial de Zea mays (L.) e Bidens pilosa (L.). Rev. Bras. Agroecol. 2013, 8, 48–56. [Google Scholar]
Galon, L.; Mossi, A.J.; Junior, F.W.R.; Reik, G.G.; Treichel, H.; Forte, C.T. Biological weed management-a short review. Rev. Bras. Herb. 2016, 15, 116–125. [Google Scholar] [CrossRef]
Fischer, G.W.; Holton, C.S. Biology and Control of the Smut Fungi; The Ronald Press Company: New York, NY, USA, 1957. [Google Scholar]
Kimati, L.H.; Filho, A.B.; Camargo, L.E.A.; Rezende, J.A.M. (Eds.) Manual de Fitopatologia, 3rd ed.; Agronômica Ceres: São Paulo, Brazil, 1997; Volume 2. [Google Scholar]
Auld, B.A. 1997 Bioherbicides. In Biological Control of Weeds: Theory and Practical Application; Julien, M., White, G., Eds.; ACIAR Monograph: Bruce, ACT, Australia; pp. 129–134.
Charudattan, R. A Reflection on My Research in Weed Biological Control: Using What We Have Learned for Future Applications. Weed Technol. 2010, 24, 208–217. [Google Scholar] [CrossRef]
Zhanzg, W.M.; Watson, A.K. Characterization of growth and conidia production of Exserohilum monoceras on different substrates. Biocontrol Sci. Technol. 1997, 7, 75–86. [Google Scholar] [CrossRef]

Figure 1. Collection sites (red triangles) [IBGE (Instituto Brasileiro de Geografia e Estatística 2021). Design ilustration: Xavier, L. C. M. (2023)].

Figure 2. Sourgrass (Digitaria insularis) showing smut symptoms (growth reduction and formation of sori) on Anthracocystis panici-leucophaei in a field situation in Monte Carmelo, Minas Gerais (Brazil).

Figure 3. Anthracocystis panici-leucophaei. (a,b) Sori on live plants (D. insularis). (c,d) Teliospores produced on fertile hyphae forming chains while immature (SEM). (e) Verruculose teliospores (SEM); (f) Detail of teliospores production. Scale bar = 2 µm.

Figure 4. Multilocus phylogenetic tree of Anthracocystis species inferred from RAxML and Bayesian analysis based on ITS sequences. The bootstraps ≥ 70 and Bayesian posterior probabilities ≥ 0.90 are indicated above the nodes, respectively. Isolate from the study are highlighted in bold. The tree was rooted with Langdonia confusa and Triodiomyces triodiae. The type isolate was identified as “t”, isotype as “i”, and holotype as “h” during isolates identification.

Figure 5. D. insularis plants 37 days after sowing. Control on the left followed by examples of plants inoculated with Anthracocystis panici-leucophaei on the right. Inoculated plants as follows: teliospores in the soil (TS1), sporidia in the soil (ES3), teliospores on 3–4 leaves plants (TA1), sporidia on 3–4 leaves plants (EA3), teliospores on newly emerged plants (TRE1), and sporidia on newly emerged plants (ERE3).

Figure 6. Examples of impact of isolates of A. panici-leucophaei on sourgrass growth 90 days after sowing 80 days after inoculation with sporidial suspension on one pair of leaves-plants. (A) Sourgrass plants inoculated with isolate BSV2; (B) Sourgrass plants inoculated with isolate 46.

Figure 7. Host-specificity evaluation of A. panici-leucophaei. Treated plants appearance at 90 days if age, 60 days after inoculation. (A) Non-inoculated (control) wheat; (B) wheat plants inoculated with the smut fungus; (C) sorghum control; (D) sorghum inoculated with the smut fungus; (E) rice control; and (F) rice inoculated with A. panici-leucophaei.

Table 1. Species included in the host-specificity test.

Species	Commom Name
Cynodon spp. cv. Tifton 85	Bermuda grass
Eleusine indica	Yard-grass
Oryza sativa	Rice
Panicum maximum cv. BRS Zuri	Guinea grass
Panicum maximum cv. híbrida BRS Quênia	Guinea grass
Panicum maximum cv. Massai	Guinea grass
Panicum maximum cv. Mombaça	Guinea grass
Panicum maximum cv. Tanzânia	Guinea grass
Pennisetum glaucum	Millet
Saccharum officinarum	Sugarcane
Sorghum bicolor	Sorghum
Triticum spp.	Wheat
Urochloa brizantha	Grass
Urochloa brizantha cv. Marandu	Grass
Urochloa brizantha cv. MG-4	Grass
Urochloa brizantha cv. MG-5 Vitória	Grass
Urochloa brizantha cv.BRS Piatã	Grass
Urochloa cv. Cayana	Grass
Urochloa híbrida cv. Sabiá	Grass
Urochloa ruziziensis cv Ruziziensis	Grass
Zea mays	Corn

Table 2. A. panici-leucophaei isolates obtained from survey in the states of Minas Gerais (MG) and Goias (GO), Brazil.

Isolate	Locality	State	Date
BSV1	Monte Carmelo	MG	21 April
BSV2	Monte Carmelo	MG	21 June
3.1	MG-223, Romaria	MG	21December
11.1	Patrocínio	MG	21 December
15.1	Ibiá	MG	21 December
16.1	Ibiá	MG	21 December
30	Carmo do Paranaíba	MG	21 December
31	BR-354—Patos de Minas	MG	21 December
36.1	São João da Serra Negra, Patrocínio	MG	21 December
37.1	Patrocínio	MG	21 December
46	Tupaciguara	MG	22 January
47	Tupaciguara	MG	22 January
49	Centralina	MG	22 January
57	Rio Verde	GO	22 January
64	Rio Verde	GO	22 January
65	Rio Verde	GO	22 January

Table 3. Effect of teliospores and sporidia application on Anthracocystis panici-leucophaei at different phenological stages of D. insularis.

Treatments	PH	RL	SD	DRM	DAPM	Clo A	NL
Control	19.48 a	24.92 a	1.98 a	0.0786 a	0.1436 a	24.16 a	4.50 bc
	PRE-EMERGENCE
TS1	7.70 b	17.07 b	1.93 a	0.0131 b	0.0754 b	6.38 c	5.35 a
ES3	9.14 b	17.64 b	1.08 b	0.0159 b	0.0621 bc	11.01 b	4.80 ab
	NEWLY EMERGED (10 DAS)—2 leaves
TRE1	7.55 b	15.37 b	1.28 b	0.0167 b	0.0380 cd	11.17 b	3.95 c
ERE3	6.82 b	14.72 b	1.07 b	0.0150 b	0.0278 d	10.47 b	4.20 bc
	POST-EMERGENCE (25 DAS)—3 to 4 leaves
TA1	7.38 b	19.12 b	1.28 b	0.0107 b	0.0521 bcd	10.40 b	4.80 ab
EA3	7.67 b	18.21 b	1.42 b	0.0103 b	0.0516 bcd	12.41 b	4.70 b
CV%	37.28	25.66	26.54	93.31	41.88	33.81	13.93

DAS: days after sowing; PH: plant height (cm); RL: root length (cm); SD: stem diameter (cm); DRM (g): dry root mass; DAPM (g): dry aerial part mass; Chlorophyll A: Chlorophyll A; NL: number of leaves. CONTROL: control group; TS1: teliospores in soil; ES3: sporidia in soil; TRE1: teliospores in newly emerged plants; ERE3: sporidia in newly emerged plants; TA1: teliospores in aerial part; EA3: sporidia in aerial part. Means followed by the same letter do not differ statistically from each other by Tukey’s test at 5% probability. Letter [a] follows means for the highest number, then b, then, c.

Table 4. Effect of A. panici-leucophaei sporidia and teliospores on D. insularis inoculated in newly emerged plants (2–3 leaves) and evaluated at 102 DAS.

Treatments	PH	RL	SD	NP	NI	NPC	DRM	DAPM	NF
CONTROL	1.14 a	0.428 a	2.85 a	3.00 ab	0.56 a	0.00 b	1.090 a	5.553 a	12.4 a
TRE	1.04 a	0.336 b	2.43 b	2.36 b	0.12 b	0.00 b	0.633 b	3.088 b	10.2 ab
BSV1	1.05 a	0.334 b	2.45 ab	2.72 ab	0.36 ab	0.00 b	0.747 b	3.485 b	10.9 a
BSV2	0.68 b	0.327 b	2.40 b	3.56 a	0.20 ab	2.16 a	0.373 c	2.703 b	8.6 b
BSV1 + BSV2	1.11 a	0.428 a	2.56 ab	2.76 ab	0.36 ab	0.00 b	0.714 b	3.164 b	11.8 a
CV%	19.86	25.54	20.24	38.11	152.09	201.28	38.13	51.47	26.61

PH: plant height (m); RL: root length (m); SD: stem diameter (cm); NP: number of tillers; NI: number of inflorescences; NPC: number of tillers with smut (sori); DRM (g): dry root mass; DAPM (g): dry aerial part mass; NF: number of leaves. TEST: control; TRE: teliospores on newly emerged plants; BSV1: sporidia of isolate BSV1 on newly emerged plants; BSV2: sporidia of isolate BSV2 on newly emerged plants; and BSV1 + BSV2: sporidia from the mixture of the two isolates on newly emerged plants. Means followed by the same letter are not statistically different by Tukey’s test at 5% probability. Letter [a] follows means for the highest number, then b, then, c.

Table 5. Comparative virulence of 16 isolates of A. panici-leucophaei to newly emerged plants of Digitaria insularis.

Treatment	PH	RL	SD	NF	NP
Treatment	cm		mm p	Leaves Plant⁻¹	Tillers Plant⁻¹
3-1	45.13 b *	30.95 c	2.32 b	8.30 a	1.52 b
11-1	41.11 b	30.77 c	1.75 b	4.94 a	0.52 a
15-1	40.62 b	24.96 b	2.01 b	10.60 b	¹ 2.52 b
16-1	55.24 c	30.45 c	2.08 b	10.22 b	1.94 b
30	45.29 b	23.35 b	1.76 b	7.00 a	1.08 a
31	34.09 a	25.75 b	¹ 1.15 a	7.26 a	0.56 a
36-1	37.86 b	27.94 c	1.90 b	8.52 a	1.92 b
37-1	34.36 a	22.10 b	¹ 1.42 a	7.52 a	0.54 a
46	24.72 a	¹ 11.36 a	¹ 1.21 a	5.12 a	1.72 b
47	40.55 b	29.81 c	1.56 a	8.62 a	1.30 a
49	41.01 b	22.25 b	¹ 1.14 a	9.62 b	1.34 a
57	44.29 b	23.43 b	1.84 b	7.34 a	0.84 a
64	39.11 b	29.66 c	1.68 a	7.56 a	1.46 b
65	45.57 b	33.03 c	2.18 b	8.46 a	1.90 b
BSV1	59.61 c	24.41 b	2.21 b	¹ 12.66 b	2.28 b
BSV2	26.01 a	29.04 c	1.56 a	6.50 a	1.14 a
Control ¹	44.58 b	29.02 c	2.26 b	6.78 a	1.14 a
CV (%)	26.11	18.21	21.47	25.76	45.60
Normality ²	0.99 ^ns	0.98 ^ns	0.99 ^ns	0.99 ^ns	0.97 ^ns
Homogeneity ³	19.55 ^ns	20.83 ^ns	0.82 ^ns	23.92 ^ns	16.50 ^ns

* Means followed by different letters in the column are statistically different according to the Scott-Knott grouping test (p < 0.05). ¹ Treatment differs from the control by the Dunnett test (p < 0.05). ² W value of the Normality test of the residuals by the Shapiro–Wilk test (p < 0.05). ³ Chi-squared value of the homogeneity test of variances by the Bartlett test (p < 0.05). Probability of the homogeneity test of variances by the Levene test for median (p < 0.05). ns, not significant. PH, plant height; RL, root length; SD, stem diameter; NF, number of leaves; NP, number of tillers. Letter [c] follows means for the highest number, then b, then, a.

Table 6. Evaluation of dry aerial part mass (DAPM), dry root mass (DRM), panicles with smut (sori), and panicles with inflorescence on mature Digitaria insularis treated with 16 isolates of Anthracocystis panici-leucophaei.

Treatment	DAPM	DRM	Smut	Inflorescence
Treatment	g Plant⁻¹		Sori Plant⁻¹	Inflorescence Plant⁻¹
3-1	1.1316 b	0.3302 b	¹ 0.34 c	0.94 b
11-1	¹ 0.4701 a *	0.1310 a	0.24 c	0.34 a
15-1	1.0352 b	0.2434 a	¹ 0.70 b	0.54 b
16-1	0.9890 b	0.2058 a	¹ 0.66 b	0.76 b
30	¹ 0.7772 a	0.1386 a	0.06 d	0.94 b
31	¹ 0.3944 a	0.1137 a	0.18 c	0.26 a
36-1	0.7848 a	0.2545 b	¹ 0.90 d	0.40 a
37-1	¹ 0.5966 a	0.2000 a	¹ 0.50 b	0.28 a
46	¹ 0.3053 a	0.1640 a	¹ 0.26 c	¹ 0.10 a
47	¹ 0.3905 a	0.1602 a	¹ 0.74 b	0.34 a
49	1.0904 b	0.1999 a	¹ 1.46 a	0.48 a
57	1.3462 b	0.2854 b	¹ 1.22 a	0.90 b
64	1.5536 c	0.5042 b	¹ 1.36 a	0.82 b
65	0.8700 b	0.2650 b	¹ 0.30 c	0.42 a
BSV1	2.0834 c	0.3914 b	0.04 d	0.92 b
BSV2	¹ 0.4476 a	0.1228 a	¹ 0.42 b	0.24 a
Control ¹	1.6156 c	0.2666 b	0.00 d	0.68 b
CV (%)	24.20	26.23	22.07	25.57
Nomality ²	0.97 ^ns	0.98 ^ns	0.98 ^ns	9.97 ^ns
Homogeneity ³	³ 21.58 ^ns	³ 20.32 ^ns	⁴ 0.74 ^ns	⁴ 0.70 ^ns

* Means followed by different letters in the column are significantly different according to the Scott-Knott grouping test (p < 0.05). ¹ Treatment differs from the control by Dunnett’s test (p < 0.05). ² W value of the Normality test of residuals by the Shapiro–Wilk test (p < 0.05). ³ Chi-squared value of the homogeneity test of variances by Bartlett’s test (p < 0.05). ⁴ Probability of the homogeneity test of variances by Levene’s test for median. ns, not significant. DAPM, dry aerial part mass; DRM, dry root mass. Letter [c] follows means for the highest number, then b, then a, for DAPM. Letter [b] follows means for the highest number, then a, for DRM. Letter [a] follows means for the highest number, then b, then c, then d, for sori plant⁻¹. Letter [b] follows means for the highest number, then a, for inflorescence plant⁻¹.

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Souza, A.P.d.; Alves, J.F.; Inokuti, E.M.; Garcia, F.; Ferreira, B.W.; Nobrega, T.F.d.; Barreto, R.W.; Vieira, B.S.; Moreira, C.C. Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis. Agronomy 2024, 14, 2926. https://doi.org/10.3390/agronomy14122926

AMA Style

Souza APd, Alves JF, Inokuti EM, Garcia F, Ferreira BW, Nobrega TFd, Barreto RW, Vieira BS, Moreira CC. Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis. Agronomy. 2024; 14(12):2926. https://doi.org/10.3390/agronomy14122926

Chicago/Turabian Style

Souza, Adriany Pena de, Juliana Fonseca Alves, Eliane Mayumi Inokuti, Fernando Garcia, Bruno Wesley Ferreira, Thaisa Ferreira da Nobrega, Robert Weingart Barreto, Bruno Sérgio Vieira, and Camila Costa Moreira. 2024. "Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis" Agronomy 14, no. 12: 2926. https://doi.org/10.3390/agronomy14122926

APA Style

Souza, A. P. d., Alves, J. F., Inokuti, E. M., Garcia, F., Ferreira, B. W., Nobrega, T. F. d., Barreto, R. W., Vieira, B. S., & Moreira, C. C. (2024). Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis. Agronomy, 14(12), 2926. https://doi.org/10.3390/agronomy14122926

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