Introduction

Weeds compete with crops for space, water, nutrients, and sunlight, causing substantial losses in crop production. Weed control in agricultural areas can be performed in a variety of ways, such as chemical, physical, cultural and biological controls. The chemical control method has been the main tool elsewhere, mainly due to excellent control effect, labor saving and quick operation1. However, many chemical pesticides because of potential human health risks, environmental pollution, the emergence of weed resistance, effects on non-target organisms, have been or being phased out2,3.

Elsholtzia densa Benth. (Labiatae: Elsholtzia), lanceolate leaves, slender inflorescences, broad-leaved weed, is widespread in areas with subtropical climate Eurasia. It grows mainly in the north of China in Hebei, Shanxi, QTP(the Qinghai-Tibetan Plateau) and adjacent areas, including Gansu, Qinghai, and Tibet, West Sichuan, etc. and threatens the productivity of crops. In Qinghai, the frequency of E. densa occurrence in spring rapeseed fields reaches 90.1%, infesting an area totaling 0.06 km24,5. Due to the limited diversity of herbicide options in rapeseed cultivation, structurally similar chemical herbicides are repeatedly applied, leading to severe herbicide resistance in E. densa.

Avena fatua (Poaceae: Avena), commonly known as wild oat, is annual grass weeds throughout the warm and cold zone of Europe, Africa, as well as many provinces of China and North America6. In cropland, this grass weed devastate crop yield by utilizing the available water before the crop mature7. Reproductive rate, intensity, frequency have increased dramatically with the expansion of annual grass weed infestation. To the farmers, these two weeds have proved a challenge, because above conventional methods have failed to suppress their growth and prevent their unchecked spread throughout the world. Activities aimed at sustainable agriculture favor the introduction of other methods to control weed infestation. Therefore, the search for new weed management strategies with safer toxicological and environmental profiles and with new modes of action has been increased8,9.

An alternative to the intensive use of chemical herbicide are the utilization of plant pathogen10. Since 1979, considerable progress has made towards practical use of plant pathogens as safe and selective agents of weed management11. Siddiqui et al.12 demonstrated that applying a solution of Alternaria alternata at a concentration of 107 spores/mL to Chenopodium album during the 2–3 leaf stage and the 4–5 leaf stage led to a significant 90% reduction in biomass. Wang13 applied a suspension of Alternaria alternata SC-018 at a spore concentration of 2.0 × 106 spores/mL to Sagittaria trifolia during the 1–2 leaf stage, resulting in over 90% mortality of the plants. Abbasi et al.14 inoculated Medicago sativa seedlings with a suspension of Alternaria alternata containing 104–105 spores/mL. Five days later, necrotic lesions appeared on the leaves and stems, ultimately leading to plant mortality. Necajeva et al.15 inoculated Echinochloa crusgalli seeds with F. culmorum at a spore concentration of 2.5 × 105 spores/mL. After 42 days, the inhibition rate of seed germination reached 73.3%. Motlagh16 inoculated 2–3 leaf-stage Echinochloa oryzicola with F. equiseti spores at a concentration of 1.0 × 105 spores/mL. The initial leaves exhibited severe spotting, ultimately leading to the death of the plants.

Some studies have demonstrated that spore/toxins produced by phytopathogens express herbicidal activity17. Alternaria sp. is one of important microorganism used for producing spore or metabolites with biological control of weeds, causing naturally lesion on leaves and stalks, as well as wilting of the plant18. Fusarium species are one of the largest genera of fungi that cause various disease such as root rot, stem blight, and spike mold on crops19. Many studies in the scientific literature demonstrated that strains of pathogenic F. oxysporum have also been selected as potential biological control agents and mycoherbicides to control and manage various parasitic weeds by destroying the tissues20.

Although these two strains have been extensively reported as biocontrol agents controlling weeds, there is still a lock of information on the herbicidal activity against E. densa and A. fatua. Take into account this context, the aim of the present study is to isolate and identify fungal pathogens naturally occurring on diseased weeds in northwest China and assess their potential for weed management.

Results

Fungal isolates

From the diseased plant leaves collected from the field, a total of fifteen fungal strains were isolated, of which seven were identified as pathogenic. The seven pathogenic isolates were obtained and labeled according to their respective host plants as follows: DT-04A2, DT-14A2, DT-YSB1 and DT-XRKA from H. cucumerifolia; DT-DYLC from C. hybridum; DT-08C from A. rosea; and DT-QKBD004A from H. vulgare. Of these, five isolates belonged to the genus Alternaria, while the remaining two belonged to the genus Fusarium. Several cultures were eliminated from further consideration because they were either contaminated or failed to grow. Specifically, several incidental isolates showing a rapid growth rate on PDA plates, including two strains of Aspergillus, four strains of Penicillium, and two strains of Rhizopus, were excluded from further consideration after their initial isolation. Ultimately, the seven pathogenic strains obtained were used for subsequent experimental research.

Pathogenicity tests on detached leaves

A detached leaf bioassay was performed by treating E. densa leaves with seven isolated strains (Table 1). As shown in Table 1, the DT-XRKA isolate produced the largest lesion area (18.07 cm2) and reached a disease incidence of 86.22 ± 2.15% on E. densa leaves at seven days after inoculation. Symptoms initially appeared as brown lesions, which then expanded, causing severe chlorosis, necrosis, and complete death of the E. densa leaves. Symptoms caused by DT-YSB1, DT-QKBD004A, DT-08C, DT-14A2, and DT-04A2 were observed sporadically at seven days, with less than one-third of the leaf area exhibiting lesions (disease grade of 2). The inoculation sites were covered with brown hyphae, and the leaves appeared wilting and curling, with an incidence rate of 54.42 ± 1.88% and a disease grade of 4 after inoculation with the DT-DYLC strain.

Table 1 Disease incidence of seven isolated strains on detached leaves of Elsholtzia densa.
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A detached leaf bioassay was performed by treating A. fatua leaves with seven isolated strains (Table 2). As shown in Table 2, the DT-QKBD004A isolate produced the largest lesion area (3.54 cm2) and reached a disease incidence of 88.06 ± 0.94% on A. fatua leaves at seven days after inoculation. Inoculated leaves exhibited progressive chlorosis, followed by the formation of expanding brown lesions that led to wilting, necrosis, and eventual death of the A. fatua leaves. Symptoms caused by DT-YSB1, DT-04A2, DT-DYLC, and DT-XRKA were observed sporadically at seven days, with a lesion area of less than 2 cm2 (disease grades of 2 and 3). In contrast, symptoms induced by DT-08C and DT-14A2 consistently manifested at seven days after inoculation, with lesions covering half to two-thirds of the leaf surface, leading to disease incidences of 63.02 ± 1.19% and 64.62 ± 0.91%, respectively (disease grade of 4).

Table 2 Disease incidence of seven isolated strains on detached leaves of Avena fatua.
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Pathogenicity tests on weed seedlings

Seven isolated strains induced symptoms in E. densa seedlings seven days post-inoculation, whereas the control treatment remained healthy (Fig. 1; Table 3). After spraying the spore suspension of the isolated strain DT-XRKA for seven days, the leaf tips of the plants wilted and curled compared to the control, and the lesion area gradually expanded, with more than three-quarters of the leaves exhibiting lesions. The plant growth was severely inhibited, with an incidence rate of 88.97 ± 2.65% and an effect on fresh weight of 85.99 ± 1.70%. Initial disease symptoms appeared with the spore suspension of DT-14A2 at three days, causing chlorosis and wilting, with about two-thirds of the leaves dying and the plant growth was severely inhibited, with an incidence rate of 80.54 ± 2.73% and a fresh weight effect of 73.90 ± 1.09% at seven days. After spraying DT-DYLC for seven days, the upper leaves of the plants began to wither and curl gradually, with the incidence gradually moving to the lower leaves; two-thirds of the leaves died, and the plant growth was severely inhibited, with an incidence rate of 69.38 ± 2.83%. When E. densa seedlings were treated with the spore suspensions of DT-08C and DT-QKBD004A, curled and wilted leaves were gradually expanding, causing one-half of the plant leaves to die. DT-04A2 and DT-YSB1 initially caused spots, followed by more than half of the E. densa seedlings exhibiting lesions, which resulting in inhibited plant growth. After comparison, strain DT-XRKA showed a significantly higher incidence rate and fresh weight control effect than the remaining strains, indicating its potential for further testing.

Fig. 1
figure 1

Pathogenicity of seven isolated strains to living potting of Elsholtzia densa. (A) Strain DT-XRKA. (B) Strain DT-14A2. (C) Strain DT-DYLC. (D) Strain DT-08C. (E) Strain DT-QKBD004A. (F) Strain DT-04A2. (G) Strain DT-YSB1. (H) Control.

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Table 3 Pathogenicity of spore suspensions of seven isolated strains to Elsholtzia densa.
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Seven isolated strains induced symptoms in A. fatua seedlings seven days post-inoculation, whereas the control treatment remained healthy (Fig. 2; Table 4). After applying the spore suspension of the isolated strain DT-QKBD004A, significant plant wilting and death were observed after seven days. Compared to the control group, more than three-quarters of the leaves exhibited necrotic lesions, and the disease symptoms were most pronounced in these plants, resulting in a disease incidence rate of 81.67 ± 1.06%. The effect on fresh weight effect was measured at 82.47 ± 1.16%. After applying the isolated strains DT-14A2 and DT-08C for seven days, significant stunting of plant height was observed, along with wilting and mortality of the lower and middle leaves. More than two-thirds of the leaves showed necrotic lesions, leading to disease incidence rates of 68.13 ± 2.41% and 67.99 ± 2.63%, respectively. Applying the isolated strains DT-DYLC, DT-XRKA, and DT-04A2 for three days resulted in scattered necrotic lesions on the upper leaves, primarily at the tips, along with stunted growth. After seven days, more than one-quarter of the plants exhibited wilting, with disease incidence rates of 45.86 ± 2.53%, 44.77 ± 1.74%, and 41.85 ± 1.91%, respectively. Strain DT-YSB1 showed scattered necrotic lesions exclusively on the upper leaves, with a disease incidence rate of 11.43% and a fresh weight effect of 11.43 ± 2.57%. Comparative analysis indicated that strain DT-QKBD004A demonstrated significantly greater effectiveness in managing both the incidence rate in A. fatua and the fresh weight effect control compared to the other strains, suggesting that this strain warrants further investigation.

Fig. 2
figure 2

Pathogenicity of seven isolated strains to living potting of Avena fatua. (A) Strain DT-QKBD004A. (B) Strain DT-14A2. (C) Strain DT-08C. (D) Strain DT-DYLC. (E) Strain DT-XRKA. (F) Strain DT-04A2. (G) Strain DT-YSB1. (H) Control.

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Table 4 Pathogenicity of spore suspensions of seven isolated strains to Avena fatua.
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Effect of different concentration of strain DT-XRKA on E. densa seedlings

Pathogenicity of strain DT-XRKA was evaluated under greenhouse conditions, where four different concentrations of spore suspensions were applied to E. densa seedlings (Fig. 3; Table 5). Symptoms were observed on the seedlings of E. densa at seven days after inoculation, with the strongest pathogenicity observed at a concentration of 1.47 × 104 spores/mL, while the control treatments remained healthy. Symptoms exhibited by DT-XRKA on E. densa were first observed at three days, which consisted of chlorisis, wilting, drying, and death after seven days. These symptoms became increasingly severe as the spore concentration increased. The percent incidence, disease index, and percent fresh weight of the inoculated E. densa seedlings with a spore suspension concentration of 1.47 × 104 spores/mL from DT-XRKA reached 96.42 ± 0.86%, 97.97 ± 1.47, and 82.67 ± 4.70%, respectively, at seven days, which were significantly higher than those of the other treatments.

Fig. 3
figure 3

Effect of different concentrations of spore suspensions from strain DT- XRKA on Elsholtzia densa (at seven days). (A) Control. (B) 1.47 × 104 spores/ml. (C) 1.47 × 103 spores/ml. (D) 1.47 × 102 spores/ml. (E) 1.47 × 101 spores/ml.

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Table 5 Pathogenicity of different spore suspension concentration of strain DT-XRKA to Elsholtzia densa.
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Effect of different concentration of strain DT-QKBD004A on A. fatua seedlings

The pathogenicity of strain DT-QKBD004A was evaluated under greenhouse conditions, where four different concentrations of spore suspension were applied to A. fatua seedlings (Fig. 4; Table 6). Symptoms induced by DT-QKBD004A on A. fatua manifested as early as two days after inoculation, characterized by chlorosis, wilting, desiccation, and mortality by seven days. The severity of these symptoms escalated with increasing spore concentration. At a spore suspension concentration of 9.40 × 104 spores/mL from DT-QKBD004A, the percent incidence, disease index, and percent fresh weight of A. fatua seedlings were measured at 89.48 ± 0.23%, 88.53 ± 1.39, and 88.42 ± 0.22%, respectively, at seven days, demonstrating significantly higher levels than those observed in the other treatments.

Fig. 4
figure 4

Effect of different concentrations of spore suspensions from strain DT-QKBD004A on Avena fatua (at seven days). (A) Control. (B) 9.40 × 104 spores/ml. (C) 9.40 × 103 spores/ml. (D) 9.40 × 102 spores/ml. (E) 9.40 × 101 spores/ml.

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Table 6 Pathogenicity of different spore suspension concentration of strain DT-QKBD004A to Avena fatua.
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Host range test of strain DT-XRKA on tested plant

Among the twelve plant species belonging to six families, including rapeseed [Brassica napus (L.)], wheat [Triticum aestivum (L.)], barley [Hordeum vulgare (L.)], broad bean [Vicia faba (L.)], pea [Pisum sativum (L.)], maize [Zea mays (L.)], pakchoi [Brassica campestris (L.)], tomato [Solanum lycopersicum (L.)], cucumber [Cucumis sativus (L.)], spinach [Spinacia oleracea (L.)], pepper [Capsicum annuum (L.)], and radish [Raphanus sativus (L.)], were used for testing (Fig. 5; Table 7). Abundant large and expanding lesions, causing more than 53% disease incidence occurred on susceptible spinach and radish when inoculated with strain DT-XRKA, with a rating of SS. The strain DT-XRKA was moderately pathogenic to Wheat, barley, broad bean and pea, with the percent incidence up to 10%-30%, with a grade of MS. Maize were slightly susceptible, showing sporadic spots on leaves and an incidence of 5.76%, with a rating of LS. No disease symptoms were observed on rapeseed, pakchoi, tomato, cucumber and pepper, with a grade of NS.

Fig. 5
figure 5

Effect of strain DT-XRKA spore suspension on crop safety. (A) Brassica napus ‘Qingza 5’ from control (left) and treatment (right). The same below. (B) Triticum aestivum ‘Qingchun 38’. (C) Hordeum vulgare ‘Kunlun 18’. (D) Vicia faba ‘Qinghai 9’. (E) Pisum sativum ‘Caoyuan 224’. (F) Zea mays ‘Qingyu 517’. (G) Brassia campestris ‘Zhebai 6’. (H) Solanum lycopersicum ‘Boyu 368’. (I) Cucumis sativus ‘Zhongnong 5’. (J) Spinacia oleracea ‘Huabo 1’. (K) Capsicum annuum ‘Zhongjiao 4’. L: Raphanus sativus ‘Chunbai 2’.

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Table 7 Effect of strain DT-XRKA spore suspension on the incidince rate and disease index of crop.
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Host range test of strain DT-QKBD004A on tested plant

Inoculation with strain DT-QKBD004A caused extensive lesions on susceptible rapeseed, resulting in over 75% disease incidence, with a rating of SS (Fig. 6; Table 8). Strain DT-QKBD004A exhibited moderate pathogenicity toward barley, spinach, and radish, with disease incidence reaching 12.71%-30.89%, with a rating of MS. Wheat and pea were slightly susceptible, showing slight curling on the leaves and an incidence of 7.62%-7.81%, with a rating of LS. No disease symptoms were observed on broad bean, maize, pakchoi, tomato, cucumber, and pepper, with a grade of NS.

Fig. 6
figure 6

Effect of strain DT-QKBD004A spore suspension on crop safety. (A) Brassica napus ‘Qingza 5’ from control (left) and treatment (right). The same below. (B) Triticum aestivum ‘Qingchun 38’. (C) Hordeum vulgare ‘Kunlun 18’. (D) Vicia faba ‘Qinghai 9’. (E) Pisum sativum ‘Caoyuan 224’. (F) Zea mays ‘Qingyu 517’. (G) Brassia campestris ‘Zhebai 6’. (H) Solanum lycopersicum ‘Boyu 368’. (I) Cucumis sativus ‘Zhongnong 5’. (J) Spinacia oleracea ‘Huabo 1’. (K) Capsicum annuum ‘Zhongjiao 4’. (L) Raphanus sativus ‘Chunbai 2’.

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Table 8 Effect of strain DT-QKBD004A spore suspension on the incidince rate and disease index of crop.
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Identification of strain DT-XRKA

Morphologic observation

On PDA medium, the colonies exhibit a sparse morphology with a rugged, filamentous texture, appearing white with a centrally located brownish hue (Fig. 7A). The hyphae were grayish-white in color, septate, and exhibited a branching pattern (Fig. 7B). The conidia displayed a claviform morphology, showing a brown coloration and measuring between 10–30 μm in length and 2–6 μm in width (Fig. 7C). Based on morphological characteristics, the strain DT-XRKA was preliminarily identified as belonging to the genus Alternaria.

Fig. 7
figure 7

Morphological characteristics of isolated strain DT-XRKA. (A) Colony. (B) Conidiophore. (C) Spore.

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Molecular identification

A further identification was conducted based on specific DNA markers frequently reported, such as ITS and Alt a1. The ITS and Alt a1 sequences of DT-XRKA were submitted to the NCBI GenBank database under accession numbers OP404082 and OP413739, respectively (Fig. 8). BLAST analysis for ITS showed homology with the species Alternaria alternata WS3 (OP161643.1) and Alternaria alternata S1 (ON827299.1), and for Alt a1 showed homology with Alternaria alternata YZU 201266 (MW308146.1). The phylogenetic tree constructed with concatenated sequences of isolate DT-XRKA showed clustering in the same clade as the sequences of Alternaria alternata. Based on morphological observation and molecular identification, strain DT-XRKA was identified as Alternaria alternata.

Fig. 8
figure 8

Phylogenetic tree of isolated strain DT-XRKA and related species constructed by neighbor-joining analyses based on the rDNA ITS (A) and Alt a1 (B) genes sequences. The serial number in brackets represents the GenBank accession number. The number on the branch point indicates the bootstrap value of the branch. The ruler represents evolutionary distances.

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Identification of strain DT-QKBD004A

Morphologic observation

On PSA medium, the colonies exhibited cottony, fluffy mycelium with a thick texture and were white in color (Fig. 9A). The hyphae were grayish-brown in color and displayed a branching pattern (Fig. 9B). The conidia were sickle-shaped and undivided, exhibiting a fawn coloration, with sizes ranging between 60–90 μm in length and 6–10 μm in width (Fig. 9C). Based on morphological characteristics, the strain DT-QKBD004A was preliminarily identified as belonging to the genus Fusarium.

Fig. 9
figure 9

Morphological characteristics of isolated strain DT-QKBD004A. (A) Colony. (B) Conidiophore. (C) Spore.

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Molecular identification

A comprehensive identification was conducted utilizing specific DNA markers frequently cited in the literature, specifically the Internal Transcribed Spacer (ITS) region and the elongation factor genes EF-1/EF-2. The corresponding ITS and EF-1/EF-2 sequences of strain DT-QKBD004A have been deposited in the NCBI GenBank database with accession numbers ON533534.1 and OP413742, respectively (Fig. 10). BLAST analysis of the ITS region revealed homology with Fusarium avenaceum SY2 (MH203387.1) and Fusarium avenaceum ITS-2ND-1-1 (OQ555101.1), while the EF-1/EF-2 sequences demonstrated homology with Fusarium avenaceum YNSF16-66 (MK185024.1) and Fusarium avenaceum 107MPT18AB (ON292377.1), with similarity reaching 100%. Analysis of the phylogenetic tree derived from concatenated sequences of isolate DT-QKBD004A revealed clustering alongside sequences of Fusarium avenaceum. Consequently, strain DT-QKBD004A was classified as Fusarium avenaceum based on morphological observations and molecular identification.

Fig. 10
figure 10

Phylogenetic tree of isolated strain DT-QKBD004A and related species constructed by neighbor-joining analyses based on the rDNA ITS (A) and EF-1/EF-2 (B) genes sequences. The serial number in brackets represents the GenBank accession number. The number on the branch point indicates the bootstrap value of the branch. The ruler represents evolutionary distances.

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Mode of infection of A. fatua by strain DT-QKBD004A

To determine whether the pathogenicity of strain DT-QKBD004A to A. fatua, detached leaves were inoculated with mycelial discs. With the prolongation of the inoculation period, the lesions at the inoculation sites on the leaves of A. fatua progressively spread (Fig. 11A). A. fatua leaves exhibited typical disease lesions, and the blue area around the inoculation site represents plant tissue cell death caused by strain DT-QKBD004A infection. As the infection time prolongs, the blue area gradually expands and progressively extends towards the edges of the leaf (Fig. 11B). In order to understand the mechanism underlying strain DT-QKBD004A pathogenicity, the histological events were monitored at different time points post-inoculation. Spores started to germinate 3 h after inoculation by producing bipolar germ tubes on the leaves. The germ tubes formed at their tips into unicellular spherical appressoria 6 h after inoculation. The appressoria started to expand at 12 h after inoculation. Extensive secondary hyphae were produced and multiplied rapidly within the leaf tissues at 15 h after inoculation, while penetration through stomata and intercellular spaces occurred at 18 h after inoculation (Fig. 11C). These results indicate that strain DT-QKBD004A causes typical necrotic lesions and establishes successful infection in A. fatua plants.

Fig. 11
figure 11

Infection mode of strain DT-QKBD004A on A. fatua leaves. (A) Following inoculation, the leaves exhibited signs of disease. (B) The leaves were stained with trypan blue solution (C) and subjected to microscopic analysis. (The red arrow indicates the appressorium structure).

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Discussion

Currently, chemical pesticides are the primary method for controlling E. densa and A. fatua in fields. However, the long-term and exclusive use of chemical herbicides often leads to increased weed resistance, shifts in weed community dynamics, reduced species diversity, decreased efficacy, and increased control costs. Sustainable weed management is a one of the main challenges for organic agriculture. Further weed management has to consider new tools, in addition to those existing, as part of integrated weed management (IWM). Mycoherbicides can help increase both the efficacy of individual weed control techniques and the overall efficacy of the IWM systems to manage weeds. Phytopathogenic fungi are one of most studies groups in relation to their herbicide potential and used to the mycoherbicides products. Mycoherbicides are products that naturally originate from either living organisms or their natural metabolites that are used to control weed populations23.

In this study, the herbicidal capacity of A. alternata DT-XRKA and F. avenaceum DT-QKBD004A as two potential biocontrol agents against E. densa and A. fatua, respectively, has been demonstrated. Based on the results of the various experiments, among 7 selected isolates, DT-XRKA and DT-QKBD004A were found to be the highest pathogenic isolates against E. densa and A. fatua. The infected plants with these two isolates showed rapid symptoms of wilting and necrotic spots on surface. 82.67% fresh weight control effect of E. densa were obtained after applying spore suspension (1.47 × 104spore/ml) produced by strain DT-XRKA. A concentration of 9.40 × 104spore/ml of strain DT-QKBD004A achieved the best fresh weight efficacy of 88.42% against A. fatua. These findings indicated that these two fungi may be useful in E. densa and A. fatua management since they affect the growth of these two weeds. Many studies also reported reduction in weeds and reproduction due to infection by fungal pathogens, reinforce the potential for pathogenic fungi to play an important role in weed management9. Damage by pathogenic fungi to E. densa and A. fatua plants results in curling of the leaves, the withering of the stem and gradual sinking of the weed.

Several studies have explored ribosomal internal transcribed spacer (ITS) to be limited and insufficient in identifying complex and variable genes and suggest the include of additional gene sequences, such as 18SrDNA, translation elongation factor 1α (TEF-1α), β-tubulin genes (TUB), etc., for the differentiation of species. In this study, the sequence data analysis the ITS, Alt a1, EF-1α along with the traditional morphological classification and the phylogenetic analysis, provided enough genetic information to reliable identification of the Alternaria and Fusarium species. Therefore, strain DT-XRKA and DT-QKBD004A were identified as Alternaria alternata and Fusarium avenaceum, respectively.

A. alternata has been described as a pathogen of water hyacinth in Australia25, Egypt26, and India27. Research conducted in these countries indicated that this fungus has potential as a biocontrol agent of water hyacinth and its toxins may also be used as an herbicide28. However, there is no report of A. alternata in E. densa control. Therefore, we consider this is one of the first studies that the control of E. densa by A. alternata. The genus of Fusarium sp. is the second genus most cited in studies related to production of mycoherbicides. Previous research evaluated the herbicidal activity of different fungi of genus Fusarium, including Fusarium avenaceum, Fusarium solani, Fusarium acuminatum, Fusarium cerealis, Fusarium redolens, in different grass species29. The authors obtained promising results when using Fusarium avenaceum and Fusarium acuminatum.

Host specificity testing and risk assessment are necessary to prevent the detrimental impacts of pathogens on non-target plants or environment30. In our study, A. alternata DT-XRKA did not infect rapeseed, cabbage, tomato, cucumber, and pepper but caused slight disease on corn, and severe disease on radish and spinach. Similarly, in another study, A. alternata caused some disease on radish and cabbage31, which indicate that these results are partially consistent with our results. F. avenaceum DT-QKBD004A was able to produce moderate to severe leaf necrosis on barley, spinach, radish, rapeseed, and did not infect on broad beans, corn, cabbage, tomato, cucumber, and pepper. Conversely, in India, a risk assessment study revealed that F. equiseti was not pathogenic to any of the crop plants test except amaranthus32. According to data of this study, 0% disease incidence were visible on rapeseed and broad bean with the inoculums of A. alternata DT-XRKA and F. avenaceum DT-QKBD004A with 104spore/ml, respectively. Thus, these two strains could be selected as mycoherbicidal candidates for rapeseed and broad bean. However, the crops safety test is only conducted at a spore concentration, and it is necessary to carry out at a series of concentrations to determine the concentration range of use for the strain and provide the basis for the application of formulation in the field.

As we know, phytopathogenic fungi are able to penetrate the leaves of plants, disintegrate their cellular structure and induce the production of necrotic lesion or chlorotic halo33. In the process of fungi infection, it involves several stages, such as attachment and germination of spore, formation of appressorium, development of infection hyphae and colonization of plant tissues34. Besides, they produce a large variety of enzymes and bioactive metabolites to facilitate host pathogenesis35.

A. alternata DT-XRKA exhibits an infection pattern similar to these previously reported Alternaria species. A. alternata DT-XRKA germinates and forms appressoria abundantly and shortly after initial germination. During this phase, hyphae penetrates through the weed tissue through leaf stomata for absorbing nutrients from the host cells, but the plant and plant cells remain alive and green, as previously observed36, and in the biotrophic phase, A. alternata DT-XRKA secrete enzymes or secondary metabolites, leading to the progressive disintegration of weed tissue and host death. Our data suggest that the biotrophic phase of A. alternata is approximately seven days long, in our test greenhouse conditions. In our detached leaf assay in a moist chamber, A. alternata-inoculated weed leaves developed disease symptoms more quickly than potted plants (data not shown), indicating the high moisture is likely important for fungal infection and development on the host.

For F. avenaceum, spore produces one or more germ tubes, which grow and expand on the host surface, and form hyphae again to invade the host tissue and absorb nutrients, which eventually lead to the death of the host tissue. These results are in good agreement with previous published reports37.

Conclusions

In this study, seven fungal pathogens were selected for evaluation of performance in detached leaf, post-emergence and crop safety assays. In these tests, A. alternata DT-XRKA and F. avenaceum DT-QKBD004A showed strong herbicidal activity on E. densa and A. fatua, respectively. Moreover, the results showed a clear host specificity of these two strains, and can be applied in rapeseed and broad bean fields, respectively. Further work on their formulation and evaluation as biocontrol agents is in progress in our lab, which may leave to recognize the potential of these two pathogens.

Materials and methods

Sites and sampling

A survey of weed-infested fields containing wheat (Triticum aestivum L.), maize (Zea mays L.), rapeseed (Brassica napus L.), peas (Pisum sativum L.), and potatoes (Solanum tuberosum L.) was conducted in multiple locations in Datong County, Xining City, Qinghai Province, China (37°07′98″N and 101°50′62″ E, altitude 3268 m). From these fields, weedy plants exhibiting visible disease symptoms were collected. The samples gathered included four types: Guayekui (Helianthus cucumerifolius L.), Hollyhock (Alcea rosea L.), Mixed quinoa (Chenopodium hybridum L.), and Hulless barley (Hordeum vulgare L.). These samples were then cut into appropriately small pieces, placed in sterilized bags, and taken to the laboratory for fungal isolation.

Isolation of the pathogen

Pathological procedures were carried out in the laboratory and greenhouse of the Key Laboratory of Agricultural Integrated Pest Management in Qinghai Province, China (36°73′07″N and 101°75′96″E, altitude 2263 m). In the laboratory, samples were superficially washed with water and dried in the open air on sterile paper. Leaf pieces of approximately 0.5 cm2 were cut from the margins of necrotic or chlorotic lesions and superficially disinfected with 75% ethanol for 1 min, followed by 1% sodium hypochlorite for 2 min, and rinsed with sterilized distilled water for 2 min. Plant tissue fragments were then placed on Petri plates containing potato dextrose agar (PDA) medium supplemented with ampicillin (1 µl/mL) under completely sterile conditions in an isolation chamber with laminar air flow. Inoculated plates were incubated at 25 °C with a 12 h light/12 h dark (12/12 h L/D) cycle and observed after seven days (seven days after inoculation). The isolates were replicated until pure colonies were obtained. The pure colonies were then transferred to fresh PDA slants and stored at 4 °C.

In vitro pathogenicity test of E. densa and A. fatua

For inoculum preparations, PDA Petri plates were inoculated by pathogenic fungi from slants and incubated at 25 °C for seven days. Each leaf of E. densa and A. fatua was surface-sterilized with 1% sodium hypochlorite (3 min) and rinsed with sterile water (5 min). For each isolate obtained, inoculation methods were evaluated by agar discs with mycelium. Two mycelial agar discs, 4 mm in diameter, were obtained directly from the growing edge of the colony and placed on each side of the midrib on the underside of leaves of E. densa and A. fatua. A control group using sterile agar discs instead of mycelial agar discs was also included. Inoculated leaves were placed on wetted filter paper (Whatman No. 4) within Petri plates, which were kept hermetically covered at room temperature (25 ± 1 °C). The incubation period, lesion area, and severity were recorded. Observations of symptom severity were performed for seven days after inoculation. The intensity of infection was determined visually, based on the initiation of disease and the increase in disease area on the leaves, every day after the application of the inoculum, using the scoring system outlined below21: Grade 1: no lesions on the leaves; grade 2: less than one-third of the leaf area having lesions; grade 3: one-third to one-half of the leaf surface covered with lesions; grade 4: one-half to two-thirds of the leaf surface covered with lesions; and grade 5: the whole leaf surface rotting or dead. Four replicates of each treatment were carried out, using each leaf as an experimental unit. The entire experiment was repeated three times independently, following the same procedure.

In vivo pathogenicity test of E. densa and A. fatua

The isolated fungi were cultured on PDA in Petri dishes and incubated for seven days at 25 °C with a 12/12 h (L/D) photoperiod. The culture surfaces on the agar plates were then harvested using 10 mL of sterilized water and a disposable cell scraper, and the harvested material was placed into test tubes containing 50 mL of sterile distilled water and 5 mL of 0.01% (v/v) Tween 80. The suspension was vortexed for 1 min to homogenize it and then filtered through sterile muslin cloth folded in four layers. The spore suspension of each fungus was counted using a hemocytometer to obtain the desired concentration of 107/mL, which was used as the inoculum source.

Weed seeds from dried and mature E. densa and A. fatua were collected from fields in 2022. The plants for the studies were grown by sowing the seeds in 15 cm diameter plastic pots containing sterilized, nutritional soil (leaf mold: garden soil: perlite: wood ash = 2: 2: 1: 1). The pots were kept in a greenhouse at 27 °C with a 12/12 h (L/D) photoperiod. Plants at the 6–8 leaf stages were sprayed with a spore suspension of the pathogens prepared as described earlier. 30 mL of a suspension of each isolate at a concentration of 1 × 106 spore/mL were applied, using a manual atomizer, until completely covering the entire aerial part of the E. densa and A. fatua plant. As a control, seedlings sprayed with sterile distilled water were used, following the same procedure described. Treated plants were placed in a greenhouse with a 12/12 h (L/D) photoperiod, at 25 °C and relative humidity above 80%, for a maximum of seven days. Disease was scored using a 1–5 point scale rating system, where 1 = no symptoms; 2 = lesions limited to the leaves; 3 = one-quarter to one-half of the leaves showing diseased spots with slight inhibition; 4 = lesions on one-half to three-quarters of the leaves with severe inhibition; and 5 = more than three-quarters of the leaf area covered by spots, leading to plant death. Each treatment was replicated four times, and the test was repeated twice. Disease incidence, disease index, and plant fresh weight efficacy on the weed plants were calculated after seven days of inoculation using the following methods:

$${\text{Disease}}\;{\text{incidence}}\; {\text{rate}}\;\left( \% \right) = \frac{{{\text{The}} \;{\text{number}}\;{\text{of}}\;{\text{diseased}}\;{\text{plants}}}}{{{\text{Total}} \;{\text{number}}\;{\text{ of}}\;{\text{plants}} \;{\text{investigated}} }} \times 100$$
$$Disease\;index = \frac{{\sum (The\;number\;of\;diseased\;plants\;at\;each\;level \times {\text{Re}} presentative\;value\;at\;each\;level)}}{{Total\;number\;of\;plants\;investigated \times {\text{Re}} presentative\;value\;at\;the\;highest\;level}} \times 100$$
$$Fresh\;weight \;efficacy\;\left(\%\right) = \frac{Plant\;fresh\;weight\;of\;control - Plant\;fresh\;weight\;of\;treated\;group}{{Plant\;fresh\;weight\;of\;control}} \times 100$$

Effect of different inoculum concentrations of fungal spore suspensions on E. densa and A. fatua seedlings

The most biocontrol potential strains obtained through preliminary screening (In vitro pathogenicity test of E. densa and A. fatua) and secondary screening (In vivo pathogenicity test of E. densa and A. fatua) were used to treat E. densa and A. fatua seedlings, diluted to spore concentrations of 101–104 spore/mL. Disease incidence, disease index, and plant fresh weight efficacy were measured at seven days. The evaluation method is the same as above.

Crop safety assessment based on optimal strains

Host-range studies were conducted on twelve plant species from various families, covering the most important field and vegetable crops in Qinghai, China. These included highland barley, wheat, rapeseed, peas, broad beans, corn, cabbage, tomato, cucumber, spinach, pepper, and white radish. All twelve crops were grown in 15 cm diameter pots and propagated from seeds. Plants at the four to five leaf stage were used in the assays, following similar inoculation and disease evaluation protocols as described above. For the control, a 0.1% Tween-80 solution in water was topically applied to each crop. After fungal inoculation, the potted plants were kept in the greenhouse under a 12/12 h (L/D) photoperiod at 25 °C with relative humidity maintained above 80%. The experiment included ten replicates per treatment and was repeated twice. The severity rating of the disease was determined as described earlier. To assess the pathogenic effect on the tested plants, a standard was used: no symptoms (NS) corresponded to 0 ≤ disease index < 5, lightly susceptible (LS) to 5 ≤ disease index < 10, moderately susceptible (MS) to 10 ≤ disease index < 50, and severely susceptible (SS) to disease index > 50.

Identification of target strains

Morphological identification

For microscopic descriptions, cultures were grown on PDA medium and incubated at room temperature (approximately 25 °C) under a 12/12 h (L/D) photoperiod for seven to fifteen days. Fungal structures were observed using an optical microscope (Nikon Eclipse E200 LED) coupled with a digital camera system (Canon EOS 700D). Aspects such as the type of mycelium, shape, diameter, and color of reproductive structures were described. For colony characterization, a 4 mm diameter mycelial disc was placed in the center of a Petri dish containing PDA medium and kept at room temperature (± 25 °C). After seven days of incubation, aspects such as texture, shape, margin, elevation of the mycelium, presence or absence of exudates, pigmentation, and color were described for both the upper and lower surfaces of the Petri dish. The information obtained was compared with fungal taxonomic keys described by Wei22.

Molecular identification

For the molecular characterization of fungal isolates, total genomic DNA was extracted using the Ezup Column Fungal Genomic DNA Purification Kit. The internal transcribed spacer (ITS) region, which encompasses the spacers between the 18S and 5.8S rRNA as well as between the 5.8S and LSU rRNA, was amplified using universal ITS primers: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Additionally, the allergen gene Alt a1 of Alternaria sp. was amplified using specific primers: Alt a1-F (5′-GCTGCACCTCTCGAGTCTC-3′) and Alt a1-R (5′-AAGTCCTTAGGGCCGTTACC-3′). Furthermore, the transcriptional elongation factor region (EF-1α) of Fusarium sp. was amplified with EF-1α primers: EF-1 (5′-ATGGGTAAGGAGGACAAGA-3′) and EF-2 (5′-CGGAAGTACCAGTGATCATG-3′). The PCR amplification system (25 μL) consisted of 12.5 μL 2 × PCR Master Mix, 1.0 μL of each primer (10 μmol/L), 1.0 μL of DNA template (25 ng/μL), and 9.5 μL of sterile double deionized water. The reaction conditions were 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 90 s, with an additional 10 min extension at 72 °C. PCR products were visualized using 1.0% agarose gel electrophoresis, and the purified products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained sequences were compared with previously reported sequences in GenBank (NCBI) using BLAST searches. Based on the results, multiple sequence alignment was performed using the Clustal W algorithm. Sequences of good quality were concatenated, and phylogenetic trees were constructed using the neighbor-joining (NJ) method implemented in MEGA 7.0 software, with each analysis performed using 1,000 bootstrap replications.

Study of the infection mechanism of the target strain on A. fatua leaves

To assess the effect of the isolated pathogen on weed leaves, inoculation assays were performed on detached weed leaves. Mycelial discs taken from a five-day-old colony of the target strain were placed in the center of the leaf surface, with PDA medium used as a negative control. The inoculated leaves were incubated in the dark at 25 °C with 80% humidity for 12, 24, 48, 72, and 96 h. The leaves were then boiled in 96% ethanol for ten minutes and stained with a trypan blue solution (lactic acid: glycerol: trypan blue: alcohol: sterilized water = 1:1:1:4:1) for two minutes, after which they were rinsed and decolorized with 96% ethanol until the background was clear.

To examine the infection process of the target strain, the process of spore germination, tube formation, and mycelial expansion in leaf tissues was observed under a light microscope. Inoculum was prepared by growing the fungus on PDA plates, and spore mass was freshly harvested by flooding the plates with sterile distilled water and then 50 µL of spore suspension (105/mL) was pipetted onto leaf surfaces using a pipettor, and the same volume of sterilized water was used as a negative control. The inoculated leaves were incubated in the dark at 25 °C with 80% humidity for 3, 6, 9, 12, 15, and 18 h. Leaf tissues around the inoculation sites were excised and also monitored by aniline blue staining (aniline blue: sterilized water = 1:100). Lesion-associated germ tubes and growth of mycelia in the leaf tissues were photographed under a light microscope. Three replicates were conducted for each treatment and the experiment was repeated two times.

Statistical analysis

Experimental data are presented as mean ± standard error, and the analysis of variance (ANOVA) was conducted on the data. Mean property values were compared using Fisher’s least significant difference (LSD) method with the SPSS package version 22.0 (p ≤ 0.05).