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Article

Infection Process of Alfalfa Root Rot Caused by Fusarium acuminatum

by
Le Wang
1,†,
Jianfeng Yang
2,†,
Ruifang Jia
1,
Zhengqiang Chen
1,
Na Wang
1,
Jie Wu
1,
Fangqi Chen
1,
Yuanyuan Zhang
1,* and
Kejian Lin
1,*
1
Key Laboratory of Biohazard Monitoring and Green Prevention and Control for Artificial Grassland, Ministry of Agriculture and Rural Affairs, Institute of Grassland Research of CAAS, Hohhot 010010, China
2
College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010018, China
*
Authors to whom correspondence should be addressed.
†
These authors equally contributed to this work.
Agronomy 2024, 14(9), 2157; https://doi.org/10.3390/agronomy14092157
Submission received: 12 August 2024 / Revised: 5 September 2024 / Accepted: 19 September 2024 / Published: 21 September 2024
(This article belongs to the Special Issue Grass and Forage Diseases: Etiology, Epidemic and Management)
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Abstract

:
Fusarium spp. can cause root rot in alfalfa, leading to the death of the whole plant, which seriously affects the yield and quality of alfalfa. This study used a Fusarium acuminatum strain labeled with green fluorescent protein (GFP) to observe the infection process of F. acuminatum on alfalfa by confocal fluorescence microscopy. The aim of this study was to reveal the infection mechanism of alfalfa Fusarium root rot at the cellular histological level. The results showed that conidia of F. acuminatum attached to the surface of the root and germinated at one day post-inoculation, the mycelium then entered the vascular bundle tissue of the alfalfa root at 5 days post-inoculation, reached the base of the plant stem at 14 days post-inoculation, and colonized the stem of the first and second compound leaf at 28 and 49 days post-inoculation, respectively. Moreover, the experiment, which sprayed a spore suspension, showed that the conidia of F. acuminatum could spread through the air to infect the pericarp and seed coat tissue of the pod. For the first time, we report the infection process of alfalfa Fusarium root rot caused by F. acuminatum and clarify that F. acuminatum can initially infect the root tissue of alfalfa, colonize the bottom stem of the plant through systematic infection, and eventually cause the plant to wilt and die. The results reveal the infection mechanism of F. acuminatum at the cell level via histology and provide theoretical support for the development of control strategies and key control technologies for alfalfa root rot.

1. Introduction

Alfalfa (Medicago sativa L.) is a globally significant perennial legume forage that is cultivated in more than 80 countries [1]. It has a higher feed protein yield per unit area compared to other forage or grain legumes. Additionally, alfalfa offers notable agronomic and environmental benefits, such as enhancing soil fertility, preventing soil erosion, fixing nitrogen annually, and reducing energy consumption and greenhouse gas emissions [2]. China has become the second largest country in terms of alfalfa planting area, with a planting area of approximately 4 million hm2. However, the adverse climate and soil conditions in major alfalfa cultivation regions in China greatly impact its yield and quality [3]. Although the statistics are incomplete, it is estimated that there are about 90 diseases affecting alfalfa in China. Of these, alfalfa root rot is the most important root disease. Fungal pathogens in the soil, particularly Fusarium spp., which belong to the Pezizomycotina subphylum, Sordariomycetes class, Hypocreomycetidae, Hypocreales order, is a significant factor affecting the growth and production of alfalfa. Alfalfa root rot caused by Fusarium leads to root destruction. The main identification points of alfalfa root rot in the field are that the whole plant is dry and dead, easy to pull up from the soil, and the root vascular bundle is brown. The development of root rot is gradual, and almost all affected plants eventually die, severely impacting forage yield and quality [4,5]. The mortality rate of alfalfa root rot exceeds 60%, with an estimated 92% of fields being seriously affected by the disease [6].
F. acuminatum is widely distributed in soil, animals, and plants [7]. Previous studies have identified F. acuminatum as the primary pathogen causing alfalfa root rot in Inner Mongolia, China [8]. Additionally, alfalfa root rot attributed to F. acuminatum has been reported in various regions worldwide [9,10,11,12]. F. acuminatum, as a significant plant pathogen, not only can cause alfalfa root rot but also infects numerous economically important crops, causing various diseases. For instance, it causes root rot and leaf blight in Dianthus chinensis [13], leaf spot in Schisandra chinensis [14], root rot in peanut (Arachis hypogaea L.) [15], leaf spot in Atradylodes lancea [16], root rot in Schisandra chinensis [17], root rot in Polygonatum odoratum [18], fruit rot in post-harvest pumpkins [19], bulb rot in garlic (Allium sativum L.) [20], and root rot in ginseng (Panax ginseng) [21], among others.
Fusarium can be transmitted in the form of mycelia and chlamydospores via intertillage weeding, irrigation water flow, and so on. In addition, Fusarium lurks inside and outside seeds in the form of mycelia, with infected seeds facilitating long-distance transmission of the disease [22]. During the whole growing season, Fusarium can infect the host at different stages of growth and re-infect the field many times. Many studies have found that some Fusarium spp. infect the root tips of plants after inoculation, and the root tip secretions induce the germination of conidia, which is conducive to the invasion of the germ tube at random sites in the root cap or root hair area [23,24,25]. The mycelium of Fusarium secretes various cell wall degrading enzymes (CWDEs) to degrade the plant cell wall, thereby facilitating invasion and colonization. At present, the CWDEs that have been found mainly include cellulase, hemicellulose, pectinase, xylitol, ligninolytic enzyme and exo-a-1, 4-galacturonase [26,27]. In addition, some pathogens can secrete heterogeneous toxins, thereby changing the permeability of the host plant cell membrane, leaking electrolytes, reducing reactive oxygen content, inhibiting the growth of plant roots, and preventing the synthesis of ATP [28,29,30].
In the study of the molecular biology of filamentous fungi, green fluorescent protein (GFP) is widely used as a naturally fluorescent protein. GFP has been widely used in plant molecular biology research, such as being used as a new reporter gene in plant transgenic engineering [31]. As a new tool of plant cell biology, it has been successfully applied in the study of the cytoskeleton, organelle dynamics, inner membrane transport, macromolecular transport, and virus movement in plants [32]. In the cloning and functional identification of unknown new genes, it is convenient to track and mark the target gene [33]. Using GFP as a reporter gene to study the interaction between plants and microorganisms, we can monitor the process of pathogen infection and related molecular mechanisms [34]. At present, many scholars have used GFP-tagged transformants of pathogenic fungi to study the infection process of pathogenic fungi on hosts. Namisy et al. [35] studied the infection progression and colonization of GFP-tagged F. oxysporum f. sp. luffae genotypes in resistant (LA140) and susceptible (LA100) luffa. Liu et al. [36] studied the colonization process of GFP-tagged Verticillium dahliae in eggplant. Cui et al. [37] used Colletotrichum coccodes tagged with GFP to observe the infection process in the Solanum tuberosum stem. Gai et al. [38] used EGFP-labeled F. vertricillioides to observe its infection process in maize stems and ears. Xu et al. [39] used GFP-labeled Pyricularia oryzae Cav. to observe the formation and development of its infection structure in the process of infecting rice, including spore germination, appressorium formation, infection nail formation, infection mycelium proliferation, necrotic spot formation, and the sporulation process.
Alfalfa root rot is a typical soil-borne disease. Currently, the prevention and control measures for soil-borne diseases are very limited and the effect is poor. Exploring the infection process of F. acuminatum in alfalfa is of great significance for the prevention and control of alfalfa root rot disease. In this study, a GFP-labeled F. acuminatum strain was used to infect alfalfa, and the infection, colonization, and expansion processes of F. acuminatum in alfalfa materials at different inoculation stages were observed by confocal fluorescence microscopy. The results could clarify the infection and pathogenicity process of F. acuminatum in alfalfa, reveal the infection mechanism of F. acuminatum at the level of cell histology, and provide theoretical support for the development of control strategies and key control technologies for alfalfa root rot.

2. Materials and Methods

2.1. Preparation of Fungal Strain and Its Conidia Suspension

HM29-05, a strain of Fusarium acuminatum labeled with GFP, is a strain preserved in our laboratory. Its morphology, biological properties, and pathogenicity were indistinguishable from the wild-type strain HM29. For conidia production, the HM29-05 transformants were firstly cultured on a potato dextrose agar (PDA) solid medium for 5 days at 25 °C, and mycelium plugs were subsequently transferred to a wheat bran medium for 7 days at 25 °C. Filtration with 4 layers of gauze was used to remove the mycelium from the fungal solution. The conidia were then counted, and the conidia concentration was adjusted to 1 × 107 spores·mL−1.

2.2. Plant Material and Growth Condition

Alfalfa cultivar Zhongmu No.1 was used as the inoculation plant and was identified as a highly susceptible genotype in a previous study [40]. Seeds were surface-sterilized in 75% alcohol for 30 s followed by thorough rinsing in sterile distilled water 3–5 times. To observe the infection and colonization process of F. acuminatum in alfalfa roots from 0 to 7 days after inoculation, an appropriate amount of seeds were sown in a sterile hydroponic box (32 cm length × 25.5 cm width × 6.7 cm height), which was covered with sterilized gauze. The hydroponic box was placed in an artificial climate chamber under a 16 h photoperiod, with temperatures of 24–26 °C and 60% relative humidity, for 5–7 days, with watering once a day. For observation of the systemic colonization of aboveground plant tissues by F. acuminatum, an appropriate amount of seeds were sown in pots (30 cm diameter × 40 cm height) filled with sterilized soil and grown in a greenhouse under a 16 h photoperiod with temperatures of 20–25 °C and 40% relative humidity. An adequate water supply was ensured during planting. A total of 10 pots were planted, and 10 plants were maintained in each pot.

2.3. Inoculation of Plants

Inoculation experiment used to observe the initial infection process of F. acuminatum in alfalfa roots: Based on the method of Kang et al. [41], an alfalfa petri dish–filter paper system was established. The square culture dish (10 cm length × 10 cm width) was prepared and modified. Four holes were made in each dish, and a layer of completely soaked absorbent cotton and sterilized filter paper was laid on the dish for use. We inoculated alfalfa seedlings in hydroponic pots by the root soaking method [42]. When the alfalfa seedlings in the hydroponic box grew to 1–2 true leaves, the alfalfa seedlings were gently pulled up from the box to avoid tearing the roots, and the roots were completely immersed in a freshly prepared conidial solution (1 × 107 spores·mLࢤ1) for 30 min. After that, the seedlings were placed in a disposable square dish one by one. Four seedlings were placed in each dish, and ten dishes were prepared. The dishes was positioned by wrapping it in tin paper and placing it, erected, in an artificial climate box for culturing, with an appropriate amount of sterile water added to the dish every day.
Inoculation experiment used to observe the systematic infection process of F. acuminatum in aboveground alfalfa plants: The alfalfa seedlings in the large pots were inoculated by the root irrigation inoculation method. When the alfalfa seedlings grew to a plant height of 15 cm, they were inoculated with 200 mL freshly prepared conidial solution (1 × 107 spores·mLࢤ1) into each pot, and a total of five pots were inoculated. The sterile water was used as the blank control. All pots were placed in a greenhouse under a 16 h photoperiod with temperatures of 20–25 °C and 40% relative humidity. Meanwhile, it was necessary to determine whether F. acuminatum could infect and colonize alfalfa seeds. A prepared 1 × 107 spores·mLࢤ1 spore suspension was evenly sprayed on immature spiral pods of healthy alfalfa plants, which were collected from the field.

2.4. Sampling of Different Plant Tissues

For the observation of the initial infection process of F. acuminatum in alfalfa roots: We sampled the alfalfa seedlings inoculated in a square petri dish. The roots were sampled every day until 7 days post-inoculation (dpi), and five roots (four inoculated and one mock) were taken from different culture dishes at each sampling time.
For the observation the systematic infection process of F. acuminatum in aboveground alfalfa plants: Samples of the alfalfa stem base, stems at different heights, petioles, and leaves were taken from large pots of alfalfa seedlings. These plant segments were sampled every week until 7 weeks post-inoculation (wpi). At each sampling time, five plants (four inoculated and one mock) were taken from different flowerpots. The plants were washed free of soil with deionized double-distilled water, air-dried, and used in section preparation. The pods were sampled for 14 dpi. A total of five pods were taken from the inoculated mature branches at the sampling time.

2.5. Confocal Microscopy

The entire root of the root sample was placed on a slide and then covered with a cover glass after washing with sterile water. It was observed from the root tip in the direction of the stem base. Several thin vertical or transverse sections were made from the collected tissues, including the stem base, main or lateral stems, petioles, and leaves, and the pods were dissected to obtain tissues such as the pericarp, seed coat, and cotyledon using sterilized double-edged razor blades. Each slice was immersed in a single drop of sterile water and covered with a coverslip. All the tissues were observed under a confocal laser scanning microscope (LSM 510; Zeiss, Jena, Germany) with GFP excitation at 488 nm. Digital images acquired from individual channels were merged using the Zeiss LSM image browser (version 4.2.0.121).

2.6. HM29-05 Re-Isolation from Root, Stem, Junction between Root and Stem, and Fresh Pods

To further confirm colonization with the strain HM29-05, different tissues of alfalfa were re-isolated after inoculation. The re-isolated strains were obtained by conventional tissue separation methods. After the re-isolated strain was purified, the colony morphology was observed on a PDA medium, and the mycelium and conidia were selected to observe the fluorescence intensity under a fluorescence microscope.

3. Results

3.1. GFP-Labeled Fusarium Acuminatum Mutant

F. acuminatum GFP-positive transformants were randomly selected and evaluated for growth rate, fecundity, toxin production, and pathogenicity versus the wild-type. The mutant HM29-05 was selected and used for all further studies to track pathogen progression, since there were no significant differences in growth rate, fecundity, colony morphology, and pathogenicity between this mutant and the wild-type isolate HM29 (Figure 1).

3.2. The Initial Infection Process of HM29-05 on Alfalfa Roots

The colonization of alfalfa plants by F. acuminatum progressed successively throughout the study period, starting in the root (Figure 2). At 1 dpi, it was observed that the conidia of F. acuminatum could adhere to the root surface and that most of the spores had germinated (Figure 2A). The germinated spores grew on the root surface to form slender mycelia. Some mycelia were attached to the surface of the root system. The mycelia initially adhered to the root hairs, mainly adhering to the root hair area, and then reached the root surface of the root hair area. At 2 dpi, the hyphae of F. acuminatum could enter the epidermal cells of the roots. At the penetration site, the hyphae formed an enlarged structure similar to an appressorium (Figure 2B), and some hyphae could form multiple such enlarged structures during the attempt to invade. At 3 post-inoculation, the mycelium grew rapidly and occupied a large area on the root surface. Most of the mycelia grew along the grooves of the epidermal cells or between the intercellular spaces (Figure 2C). At 4 dpi, the hyphae gradually expanded horizontally from the edge of the root system to the center and longitudinally along the intercellular space (Figure 2D). The penetration and colonization of root tissue accelerated by 5 dpi. The mycelium reached the vascular bundle tissue and grew along the xylem (Figure 2E). At 7 dpi, the mycelium began to expand horizontally or vertically in the cortical tissue, and a large number of hyphae were colonized in the vascular bundle and cortex of the root system (Figure 2F,G). No green fluorescence signal was observed in the vascular tissue of uninoculated plants roots (Figure 2H).

3.3. Systematic Infection Process of HM29-05 on Alfalfa Aboveground Plants

The stem tissues of alfalfa inoculated with F. acuminatum at different positions in pots were observed by confocal fluorescence microscopy. At 14 post-inoculation, the stem base was sampled and observed by fluorescence microscopy. It was found that obvious green fluorescence was observed in the epidermal and cortical cells of the stem base. The mycelium was mainly concentrated in the cortical cells, and a small part of the mycelium extended to the central vascular bundle (Figure 3A). In the longitudinal section, the mycelium could be observed to extend vertically in the cortical cell gap or horizontally to adjacent cells (Figure 3B). At 28 dpi, the stem part with a length of 1 cm at the first compound leaf was cut for section observation, and obvious green fluorescence could be seen in both transverse and longitudinal sections (Figure 3C,D). The mycelia could colonize and expand longitudinally in the cortex and vascular bundle tissues, but no fluorescence signal was observed in the stem at the second compound leaf. At 49 dpi, alfalfa plants showed obvious yellowing of their leaves and wilting or drying of their branches (Figure 4). The green fluorescence signal was observed in the cortex and vascular bundle tissues of the transverse and longitudinal sections of the upper part of the stem (Figure 3E,F), but no fluorescence signal was observed in the stem of the third compound leaf (Figure 3G,H).
After spraying the HM29-05 strain onto immature pods of alfalfa, the different tissues of the pods, such as the peel, seed coat, and cotyledon, were observed by fluorescence microscopy. The results showed that, 14 after inoculation, F. acuminatum had colonized the epidermal cells and cortical tissues of the peel (Figure 5A,B), indicating that the conidia of F. acuminatum could adhere to the surface of the pod and germinate. After entering the epidermal cells, it extended into the cortical tissue, but a small part of the hyphae entered the vascular bundle tissue. Only a small amount of hyphae were observed on the seed coat (Figure 5C), and no colonization of F. acuminatum was observed in the cotyledon tissue (Figure 5D).

3.4. Re-Isolation

By re-isolating different stem and pod tissues of alfalfa after inoculation, the colony morphology of the pure culture obtained was the same as that of F. acuminatum (Figure 6A,B). The selected mycelium and conidia emitted strong green fluorescence under a fluorescence microscope (Figure 6C,D), once again proving that the strain HM29-05 can infect the stem and pod tissues of alfalfa.

3.5. Infection Mode of Fusarium Acuminatum on Alfalfa

The pattern of F. acuminatum infecting alfalfa is shown in Figure 7. The initial infection site of F. acuminatum in alfalfa is at the root tip. At 1 dpi, the conidia of F. acuminatum adhered to the root surface. At 2 dpi, the hyphae had begun to invade the epidermal cells of the roots. At the penetration site, the hyphae formed an enlarged structure similar to an appressorium. At 3 post-inoculation, the mycelia grew rapidly and occupied a large area on the root surface. Most of the mycelia grew along the grooves of epidermal cells or between intercellular spaces, and a small amount of mycelia entered the cortical tissue and began to grow horizontally or vertically. At 5 dpi, the hyphae had reached the threaded catheter of the vascular bundle and began to expand horizontally or vertically. The strain HM29-05 was able to colonize the stem base of alfalfa, and a large number of mycelia colonized the cortex tissue, while a small number of mycelia entered the vascular bundle at 14 dpi. At 28 days post-inoculation, strain HM29-05 could colonize the stem of the first compound leaf of the alfalfa plant. And at 49 dpi, the inoculated alfalfa plants showed yellowing of their leaves and wilting or drying of their branches, and green fluorescence signals were detected in the stem bundles of the second compound leaf of alfalfa plants. At 14 days post-spraying, F. acuminatum could infect and colonize the seed coat and epidermal tissue of fresh pods of alfalfa.

4. Discussion

In the current study, the conidial suspension of F. acuminatum with the GFP marker was used to inoculate the susceptible variety of alfalfa ‘Zhongmu NO.1′, and the infection process of F. acuminatum in alfalfa was observed under a confocal fluorescence microscope. The results showed that the initial infection site of F. acuminatum on alfalfa was the root hair area. At 1 day post-inoculation, most of the spores attached to the root surface of the alfalfa had germinated to form germ tubes and grew in all directions on the root surface. The primary infection site was in the root hairs of the roots, which was similar to the infection pattern of F. oxysporum on different host plants, such as banana [43] and tomato [44]. Mycelia colonizing epidermal cells will form an enlarged structure similar to an appressorium at the osmotic site to improve the infection ability of pathogens to host plants. Studies have shown that the cell walls of appressoria are highly degraded, lacking in chitin and thinner than a normal cell wall in the contact area between the appressoria and plant surface and around the infection point [45]. In this study, we observed that the mycelium could form multiple such swelling structures during growth and extension, which was consistent with the results of Perry et al. [46] in observing the infection of Verticillium dahliae in potato. Studies had shown that the pre-penetration stage of F. acuminatum was mainly the extensive colonization of the root surface and that the hyphae of the fungus grew horizontally and vertically on the root surface, which may be related to the maintenance of mycelial growth and reproduction by root exudates. Parry et al. [47] reported that F. avenaceum and F. culmorum colonized on the root surface of alfalfa seedlings, but only a few hyphae were observed between the epidermal cell walls in seedlings inoculated with F. oxysporum. At 3 dpi, most hyphae grew along the grooves of epidermal cells or between intercellular spaces, which was consistent with the results reported by Bishop et al. [48] that intercellular penetration was achieved by the inward growth of hyphal tips between epidermal cells. In addition, the grooves formed between the longitudinal walls of adjacent epidermal cells may be repositories of nutrients that support the growth of microorganisms on the root surface [49]. At 5 dpi, the hyphae penetrated the cortical tissue into the vascular bundle of the alfalfa. With the passage of time, it was observed that F. acuminatum mainly proliferated in the cells around the vascular bundle. In order to analyze the infection mechanism of F. oxysporum on Rehmannia glutinosa, Liu et al. [50] observed the infection process of sGFP-labeled strains on R. glutinosa plants by the root inoculation method. The results showed that F. oxysporum did not directly invade R. glutinosa within 3 days after inoculation. Until 5 dpi, F. oxysporum was observed to invade the vascular tissue of R. glutinosa, which was similar to the initial infection mode of F. acuminatum on alfalfa roots in this study.
Through observation, it was found that, in the process of F. acuminatum infecting alfalfa, there was no wilting phenomenon in the leaves at the early stage of infection, and the typical root rot disease occurred in the late stage of plant growth. The infection process of F. acuminatum on aboveground alfalfa plants was observed by fluorescence microscopy. It was clear that F. acuminatum colonized the base of the stem of alfalfa at 14 post-inoculation. At 28 dpi, the mycelia could extend vertically upward to the first compound leaf of the stem. Until 49 dpi, F. acuminatum could reach the second compound leaf of the stem of alfalfa, but no fluorescence signal was observed in the stem of the third compound leaf, and then the whole branch withered. Studies had shown that the initial infection of F. oxysporum begins with the germination of conidia attached to the root surface, and then the mycelia penetrate the epidermal cells, the cortex, and the vascular system. Subsequently, F. oxysporum infiltrate and colonize the xylem, and hyphae colonize the vascular bundles of stems and branches vertically through the movement of xylem vessels [48,51], which is different from the infection process of F. acuminatum in alfalfa in this study. There are two theories about the pathogenic mechanism of vascular bundle diseases. One is that, after the pathogen infects and colonizes the xylem vessel of the host, a colloidal substance will be produced in the vessel, which will block the vessel and cause the host to wither. Another theory points out that pathogens can produce wilting toxins, change the permeability of the host cell membrane, destroy the function of mitochondria and chloroplasts, and cause the host to wither [52]. In this experiment, from the fluorescence observation results of transverse and longitudinal sections of different stem tissues, it can be seen that the hyphae infect the cortex tissue in the bottom stem of alfalfa plants in large quantities, and only a few hyphae colonize the central vascular bundle tissue. Therefore, further confirmation is needed to determine whether F. acuminatum causes plant wilting by blocking vascular bundle catheter tissues or secreting wilting toxins.
Due to the infection of F. acuminatum, the cortex tissue and vascular bundle system of alfalfa were damaged, and the branches were wilted and could not complete the flowering and fruiting stages. In order to explore whether F. acuminatum can infect and colonize alfalfa seeds, we sprayed a spore suspension of F. acuminatum on immature spiral pods of healthy alfalfa plants. By observing the infection process of F. acuminatum on alfalfa pods and seeds, it was found that F. acuminatum can infect and colonize the pericarp and seed coat tissues of pods, but does not appear in the internal cotyledons. Sun et al. [53] pointed out in a study of the infection process of F. proliferatum on rice that, during the flowering period, spraying the agent can cause the pathogen to expand to the endosperm surface through the endosperm lemma and palea, but the infection of the pathogen in the endosperm itself is not obvious. The results of that study are obviously inconsistent with the infection of F. acuminatum in alfalfa embryos in this paper. The reason for this phenomenon may be that the oil content in cotyledons is high, and the pathogen is difficult to colonize in this part. In addition, when the laboratory examined fungi carried by alfalfa seeds in the field, it was found that the probability of alfalfa seeds carrying Fusarium was 32% (Unpublished). This phenomenon may be caused by various environmental factors, such as temperature and humidity in the field. We consider the next step to be the observation of the infection process of Fusarium in alfalfa under field environmental conditions, and the further clarification of the source of alfalfa seeds carrying fungi. Combined with the observational results of this study, it was indicated that the spores of Fusarium in the air could attach to pods and further infect the internal seed coat. Seeds carrying fungi increase the risk of disease occurrence and an epidemic, which provides the possibility for the long-distance transmission of Fusarium root rot [54].

5. Conclusions

The infection process of GFP-labeled F. acuminatum on alfalfa was observed by confocal fluorescence microscopy, and it was clear that F. acuminatum could initially infect the root tissue of alfalfa, colonize the bottom stem of the plant through systematic infection, and eventually cause the wilting and death of the plant. In addition, the conidia of F. acuminatum could also infect and colonize the pericarp and seed coat tissue of the pod through air transmission.

Author Contributions

Conceptualization, K.L.; Methodology, J.Y. and N.W.; Validation, R.J.; Formal analysis, L.W.; Investigation, J.Y. and J.W.; Data curation, Z.C. and F.C.; Writing—original draft, L.W.; Writing—review & editing, Y.Z.; Project administration, K.L.; Funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (2022YFD1300802).

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The financial support mentioned in the Funding part is gratefully acknowledged, and thank you to all the authors for their support and help in this research.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Summers, C.G.; Putnam, D.H. Irrigated Alfalfa Management for Mediterranean and Desert Zones; ANR Publications: Oakland, CA, USA, 2008. [Google Scholar]
  2. Annicchiarico, P. Alfalfa forage yield and leaf/stem ratio: Narrow-sense heritability, genetic correlation, and parent selection procedures. Euphytica 2015, 205, 409–420. [Google Scholar] [CrossRef]
  3. Wang, T.; Zhang, W. Priorities for the development of alfalfa pasture in northern China. Fundam. Res. 2023, 3, 225–228. [Google Scholar] [CrossRef] [PubMed]
  4. Graham, J.H.; Frosheiser, F.I.; Stuteville, D.L.; Erwin, D.C. Compendium of Alfalfa Diseases; American Phytopathological Society: St. Paul, MN, USA, 1990. [Google Scholar]
  5. Guo, Y.X.; Guo, Z.P.; Zhang, J.X.; Wang, M.; Qu, G.; Yan, X.; Zhang, M. Study on the pathogenicity of Fusarium semitectum and disease resistance of alfalfa varieties. Acta Phytopathol. Sin. 2018, 48, 108–118. [Google Scholar]
  6. Fernandez, A.L.; Sheaffer, C.C.; Tautges, N.E.; Putnam, D.H.; Hunter, M.C. Alfalfa, Wildlife and the Environment; National Alfalfa and Forage Alliance: St. Paul, MN, USA, 2019. [Google Scholar]
  7. Booth, C. The genus Fusarium; Common-Wealth Mycological Institute: London, UK, 1971. [Google Scholar]
  8. Wang, L.; Wang, N.; Yu, J.; Wu, J.; Liu, H.; Lin, K.; Zhang, Y. Identification of pathogens causing alfalfa Fusarium root rot in Inner Mongolia, China. Agronomy 2023, 13, 456. [Google Scholar] [CrossRef]
  9. Hawn, E.J. Studies on the epidemiology of crown bud rot of alfalfa in southern. Can. J. Bot. 1958, 36, 239–250. [Google Scholar] [CrossRef]
  10. Uddin, W. Fusarium species associated with crown rot of alfalfa in Nevada. Plant Dis. 1991, 75, 51–56. [Google Scholar] [CrossRef]
  11. An, H.L. Study on the Pathogen of Alfalfa Root Rot in Gansu Province; Lanzhou University: Lanzhou, China, 2016. [Google Scholar]
  12. Yi, M. Identification of Pathogens of Alfalfa Root Rot and Spot Disease in Shaanxi Province; Northwest A & F University: Xianyang, China, 2016. [Google Scholar]
  13. Xu, J.; Jiao, B.; Xia, H.; Dai, T. First report of Fusarium acuminatum causing Dianthus chinensis root rot and foliage blight in China. Plant Dis. 2023, 107, 2254. [Google Scholar] [CrossRef] [PubMed]
  14. Zhan, Y.; Yan, N.; Miao, X.; Wang, E.; Zhou, Y.; Chen, C.; Qiong, L. First report of Fusarium acuminatum causing leaf spot on Schisandra chinensis in China. Plant Dis. 2023, 107, 2872. [Google Scholar] [CrossRef]
  15. Li, Y.; Yu, J.; Guo, Z.; Song, X.; Xu, M.; He, K.; Zhang, X.; Chi, Y. First report of peanut root rot caused by Fusarium acuminatum in Shandong province, China. Plant Dis. 2023, 107, 2882. [Google Scholar] [CrossRef]
  16. Zheng, Y.; Cui, Q.; Li, Q.; Tian, P.; Peng, W.; Guo, X.; Xu, S.; Yang, X.; Zhang, X.; Xie, C. First report of Atradylodes lancea leaf spot caused by Fusarium acuminatum in China. Plant Dis. 2023, 107, 968. [Google Scholar] [CrossRef]
  17. Shen, B.Y.; Sun, W.S.; Liu, K.; Zhang, J.T. First report of root rot of Schisandra chinensis caused by Fusarium acuminatum in northeast China. Plant Dis. 2022, 106, 1987. [Google Scholar] [CrossRef] [PubMed]
  18. Li, X.; Sun, W.; Shen, B.; Liu, K.; Zhang, T. First report of root rot of Polygonatum odoratum caused by Fusarium acuminatum in China. Plant Dis. 2021, 105, 4161. [Google Scholar] [CrossRef] [PubMed]
  19. Li, Y.G.; Jiang, W.Y.; Jiang, D.; Wang, R.T.; Tian, S.; Ji, P.; Jiang, B.W. First report of fruit rot on postharvest pumpkin caused by Fusarium acuminatum in China. Plant Dis. 2019, 103, 1035. [Google Scholar] [CrossRef]
  20. Ignjatov, M.; Bjelic, D.; Nikolic, Z.; Milosevic, D.; Gvozdanovic-Varga, J.; Marinkovic, J.; Ivanovic, Z. First report of Fusarium acuminatum causing garlic bulb rot in Serbia. Plant Dis. 2017, 101, 1047–1048. [Google Scholar] [CrossRef]
  21. Wang, Y.; Guan, Y.M.; Lu, B.H.; Gao, J. First report of ginseng (Panax ginseng) root rot caused by Fusarium acuminatum in China. Plant Dis. 2016, 100, 525–526. [Google Scholar] [CrossRef]
  22. Chen, Y.J.; Liu, X.M.; Cui, G.W.; Xi, Q.X. Research progress in root rot of alfalfa. Grassl. China 2000, 1, 51–56. [Google Scholar]
  23. Jia, R.; Kang, L.; Addrah, M.E.; Zhang, J.; Xu, L.; Zhang, Z.; Chen, W.; Liu, J.; Zhao, J. Potato wilt caused by co-infection of Fusarium spp. and Verticillium dahliae in potato plants. Eur. J. Plant Pathol. 2023, 165, 305–315. [Google Scholar] [CrossRef]
  24. Li, C.X.; Fu, X.P.; Zhou, X.G.; Liu, S.W.; Xia, Y.; Li, N.H.; Zhang, X.X.; Wu, F.Z. Treatment with wheat root exudates and soil microorganisms from wheat/watermelon companion cropping can induce watermelon disease resistance against Fusarium oxysporum f. sp. niveum. Plant Dis. 2019, 103, 1693–1702. [Google Scholar] [CrossRef]
  25. Yang, L.; Zhou, Y.; Guo, L.; Yang, L.; Wang, J.; Liang, C.; Huang, J. The effect of banana Rhizosphere chemotaxis and chemoattractants on Bacillus velezensis LG14-3 root colonization and suppression of banana Fusarium wilt disease. Sustainbility 2023, 15, 351. [Google Scholar] [CrossRef]
  26. Kubicek, C.P.; Starr, T.L.; Glass, N.L. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annu. Rev. Phytopathol. 2014, 52, 427–451. [Google Scholar] [CrossRef]
  27. Quoc, N.B.; Chau, N. The role of cell wall degrading enzymes in pathogenesis of Magnaporthe oryzae. Curr. Protein Pept. Sci. 2017, 18, 1019–1034. [Google Scholar] [CrossRef] [PubMed]
  28. Bian, C.; Duan, Y.; Xiu, Q.; Wang, J.; Tao, X.; Zhou, M. Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum. Mol. Plant Pathol. 2021, 22, 769–785. [Google Scholar] [CrossRef] [PubMed]
  29. Bouizgarne, B.; Brault, M.; Pennarun, A.M.; Rona, J.P.; Ouhdouch, Y.; Hadrami, I.E.; Bouteau, F. Electrophysiological responses to fusaric acid of root hairs from seedlings of date palm-susceptible and -resistant to Fusarium oxysporum f. sp. albedinis. J. Phytopathol. 2004, 152, 321–324. [Google Scholar] [CrossRef]
  30. Marrè, M.T.; Vergani, P.; Albergoni, F.G. Relationship between fusaric acid uptake and its binding to cell structures by leaves of Egeria densa and its toxic effects on membrane permeability and respiration. Physiol. Mol. Plant Pathol. 1993, 42, 141–157. [Google Scholar] [CrossRef]
  31. Hansen, G.; Wright, M.S. Recent advances in the transformation of plants. Trends Plant Sci. 1999, 4, 226–231. [Google Scholar] [CrossRef]
  32. Köhler, R.H.; Zipfel, W.R.; Webb, W.W.; Hanson, M.R. The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J. Cell Mol. Biol. 1997, 11, 613–621. [Google Scholar] [CrossRef]
  33. Haseloff, J. GFP variants for multispectral imaging of living cells. Methods Cell Biol. 1999, 58, 139–151. [Google Scholar]
  34. Bowyer, P.; Mueller, E.; Lucas, J. Use of an isocitrate lyase promoter-GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of wheat. Mol. Plant Pathol. 2000, 1, 253–262. [Google Scholar] [CrossRef]
  35. Namisy, A.; Chen, S.Y.; Huang, J.H.; Unartngam, J.; Thanarut, C.; Chung, W.H. Histopathology and quantification of green fluorescent protein-tagged Fusarium oxysporum f. sp. luffae isolate in resistant and susceptible Luffa germplasm. Microbiol. Spectr. 2024, 12, e0312723. [Google Scholar] [CrossRef]
  36. Liu, Q.; Li, Y.; Wu, H.; Zhang, B.; Liu, C.; Gao, Y.; Guo, H.; Zhao, J. Hyphopodium-specific signaling is required for plant infection by Verticillium dahliae. J. Fungi 2023, 9, 484. [Google Scholar] [CrossRef]
  37. Cui, Y.; Yang, C. Infection process of Colletotrichum coccodes tagged with GFP on Solanum tuberosum. Physiol. Mol. Plant Pathol. 2021, 115, 101665. [Google Scholar] [CrossRef]
  38. Gai, X.; Dong, H.; Wang, S.; Liu, B.; Zhang, Z.; Li, X.; Gao, Z. Infection cycle of maize stalk rot and ear rot caused by Fusarium verticillioides. PLoS ONE 2018, 13, e0201588. [Google Scholar] [CrossRef]
  39. Xu, Y.P.; Liao, H.C.; Chen, J.H.; Luo, Y.; Su, J.; Zou, C.D.; Li, W.T.; Wang, J.; Ma, B.T.; He, M.; et al. Fluorescent microscopic analysis of the plant infection process of Magnaporthe oryzae using green fluorescent protein. Plant Prot. 2017, 43, 53–61. [Google Scholar]
  40. Zhang, Y.Y.; Wang, L.; Lin, K.J.; Yue, F.Z.; Xu, L.B. Evaluation of disease resistance of different alfalfa germplasms to Fusarium root rot. Chin. J. Grassl. 2021, 43, 98–105. [Google Scholar]
  41. Kang, L.R.; Gao, J.; Jia, R.F.; Liu, Y.; Fan, J.C.; Zhang, Z.W.; Zhao, J. Utilization of GFP tagged Verticillium dahliae strain to evaluate dynamic infection on resistant/susceptible potato varieties. Acta Phytopathol. Sin. 2023, 53, 164–172. [Google Scholar]
  42. Bhat, R.G.; Subbarao, K.V. Host range specificity in Verticillium dahliae. Phytopathology 1999, 89, 1218–1225. [Google Scholar] [CrossRef]
  43. Li, C.; Chen, S.; Zuo, C.; Sun, Q.; Ye, Q.; Yi, G.; Huang, B. The use of GFP-transformed isolates to study infection of banana with Fusarium oxysporum f. sp. cubense race 4. Eur. J. Plant Pathol. 2011, 131, 327–340. [Google Scholar] [CrossRef]
  44. Molina, M.C.R.; Medina, I.; Vila, L.M.T.; Cuartero, J. Vascular colonization patterns in susceptible and resistant tomato cultivars inoculated with Fusarium oxysporum f.sp. lycopersici races 0 and 1. Plant Pathol. 2003, 52, 199–203. [Google Scholar] [CrossRef]
  45. Bourett, T.M.; Howard, R.J. In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Can. J. Bot. 1990, 68, 329–342. [Google Scholar] [CrossRef]
  46. Perry, J.W.; Evert, R.F. The effect of colonization by Verticillium dahliae on the root tips of russet burbank potatoes. Can. J. Bot. 1983, 61, 3422–3429. [Google Scholar] [CrossRef]
  47. Parry, D.W.; Pegg, G.F. Surface colonization, penetration and growth of three Fusarium species in lucerne—ScienceDirect. Trans. Br. Mycol. Soc. 1985, 85, 495–500. [Google Scholar] [CrossRef]
  48. Bishop, C.D.; Cooper, R.M. An ultrastructural study of root invasion in three vascular wilt diseases—ScienceDirect. Physiol. Plant Pathol. 1983, 22, 13–15. [Google Scholar] [CrossRef]
  49. Bowen, G.D. Integrated and experimental approaches to the study of growth of organisms around roots. In Control of Messenger RNA Stability; Academic Press: San Diego, CA, USA, 1993. [Google Scholar]
  50. Liu, X.Y.; Luo, Z.P.; Cui, W.K.; Lin, Z.F.; Yue, K.H.; Wang, S.R.; Li, Z.F. Histological observation of the infection process in Rehmannia glutinosa by Fusarium oxysporum labeled with green fluorescent protein (GFP) gene. Mycosystema 2015, 34, 1092–1100. [Google Scholar]
  51. Zvirin, T.; Herman, R.; Brotman, Y.; Denisov, Y.; Belausov, E.; Freeman, S.; Perl-Treves, R. Differential colonization and defence responses of resistant and susceptible melon lines infected by Fusarium oxysporum race 1·2. Plant Pathol. 2010, 59, 576–585. [Google Scholar] [CrossRef]
  52. Wu, G.Y. Distribution of Defoliating Pathotype and Toxicity Test of Verticillium dahliae in Shandong Province and Screening of Biocontrol Bacterium; Shandong Agricultural University: Taian, China, 2014. [Google Scholar]
  53. Sun, L.; Wang, L.; Liu, L.; Hou, Y.; Yihua, X.U.; Liang, M.; Gao, J.; Qiqin, L.I.; Huang, S. Infection and colonization of pathogenic fungus Fusarium proliferatum in rice spikelet rot disease. Rice Sci. 2019, 26, 9. [Google Scholar]
  54. Feng, S.X.; Jiang, N.; Chen, Q.P. Detection of germs in seed and effect analysis of sterilization treatment of Euchresta japonica hook. f. ex regel. Chin. J. Trop. Agric. 2022, 42, 70–75. [Google Scholar]
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Figure 1. Photographs showing biological characterization and pathogenicity of the Fusarium acuminatum wild-type strain HM29 and the mutant strain HM29-05. On the left: colony morphology of the Fusarium acuminatum wild-type strain HM29 and mutant strain HM29-05 on potato dextrose agar at 5 days; on the right: disease symptoms of the Fusarium acuminatum wild-type strain HM29 and mutant strain HM29-05 on alfalfa seedlings at 6 weeks post-inoculation.
Figure 1. Photographs showing biological characterization and pathogenicity of the Fusarium acuminatum wild-type strain HM29 and the mutant strain HM29-05. On the left: colony morphology of the Fusarium acuminatum wild-type strain HM29 and mutant strain HM29-05 on potato dextrose agar at 5 days; on the right: disease symptoms of the Fusarium acuminatum wild-type strain HM29 and mutant strain HM29-05 on alfalfa seedlings at 6 weeks post-inoculation.
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Figure 2. Infection process and colonization of Fusarium acuminatum in the alfalfa root from 1 day post-inoculation (dpi) to 7 dpi. A F. acuminatum mutant strain, HM29-05, that expressed green fluorescent protein (GFP) inoculated in a 3-day-old seedling alfalfa var. ‘Zhongmu NO.1′ by the root soaking method. (A) Germinated conidia on the root surface at 1 dpi, scale bars = 25 um; (B) the germinated spores grew on the root surface to form slender mycelia at 2 dpi, scale bars = 25 um; (C) the mycelia grew along the grooves of the epidermal cells or between the intercellular spaces at 3 dpi, scale bars = 25 um; (D) the mycelia expanded horizontally or vertically in the cortical tissue at 4 dpi, scale bars = 50 um; (E) the mycelia reached the vascular bundle tissues at 5 dpi, scale bars = 25 um; (F) more hyphae were colonized in the cortical cells and formed mycorrhizal networks at 7 dpi, scale bars = 50 um; (G) a large number of hyphae were colonized in the vascular bundle and cortex of the root system at 7 dpi, scale bars = 50 um; and (H) no green fluorescence signal in the vascular tissue of uninoculated plants roots at 7 dpi, scale bars = 25 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
Figure 2. Infection process and colonization of Fusarium acuminatum in the alfalfa root from 1 day post-inoculation (dpi) to 7 dpi. A F. acuminatum mutant strain, HM29-05, that expressed green fluorescent protein (GFP) inoculated in a 3-day-old seedling alfalfa var. ‘Zhongmu NO.1′ by the root soaking method. (A) Germinated conidia on the root surface at 1 dpi, scale bars = 25 um; (B) the germinated spores grew on the root surface to form slender mycelia at 2 dpi, scale bars = 25 um; (C) the mycelia grew along the grooves of the epidermal cells or between the intercellular spaces at 3 dpi, scale bars = 25 um; (D) the mycelia expanded horizontally or vertically in the cortical tissue at 4 dpi, scale bars = 50 um; (E) the mycelia reached the vascular bundle tissues at 5 dpi, scale bars = 25 um; (F) more hyphae were colonized in the cortical cells and formed mycorrhizal networks at 7 dpi, scale bars = 50 um; (G) a large number of hyphae were colonized in the vascular bundle and cortex of the root system at 7 dpi, scale bars = 50 um; and (H) no green fluorescence signal in the vascular tissue of uninoculated plants roots at 7 dpi, scale bars = 25 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
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Figure 3. Infection process and colonization of Fusarium acuminatum in alfalfa plants from 1 week post-inoculation (wpi) to 7 wpi. A F. acuminatum mutant strain, HM29-05, that expressed green fluorescent protein (GFP) inoculated in a 15 cm tall alfalfa plant var. ‘Zhongmu NO.1’ by the root irrigation inoculation method. (A) The colonization at the stem base from a transverse section at 14 dpi, scale bars = 75 um; (B) a few hyphae extended to the central vascular bundle at the stem base in a longitudinal section at 14 dpi, scale bars = 50 um; (C) the mycelium colonized at the first compound leaf in a transverse section at 28 dpi, scale bars = 100 um; (D) the mycelium colonized and expanded vertically in the cortex and vascular bundle tissues at the first compound leaf in a longitudinal section at 28 dpi, scale bars = 100 um; (E) the mycelium at the second compound leaf of an alfalfa plant in a transverse section at 49 dpi, scale bars = 50 um; (F) the fluorescence signal observed in the second compound leaf in a longitudinal section at 49 dpi, scale bars = 100 um; (G) no fluorescence signal was observed in the third compound leaf in a transverse section at 49 dpi, scale bars = 100 um; and (H) no fluorescence signal was observed in the third compound leaf in a longitudinal section at 49 dpi, scale bars = 250 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
Figure 3. Infection process and colonization of Fusarium acuminatum in alfalfa plants from 1 week post-inoculation (wpi) to 7 wpi. A F. acuminatum mutant strain, HM29-05, that expressed green fluorescent protein (GFP) inoculated in a 15 cm tall alfalfa plant var. ‘Zhongmu NO.1’ by the root irrigation inoculation method. (A) The colonization at the stem base from a transverse section at 14 dpi, scale bars = 75 um; (B) a few hyphae extended to the central vascular bundle at the stem base in a longitudinal section at 14 dpi, scale bars = 50 um; (C) the mycelium colonized at the first compound leaf in a transverse section at 28 dpi, scale bars = 100 um; (D) the mycelium colonized and expanded vertically in the cortex and vascular bundle tissues at the first compound leaf in a longitudinal section at 28 dpi, scale bars = 100 um; (E) the mycelium at the second compound leaf of an alfalfa plant in a transverse section at 49 dpi, scale bars = 50 um; (F) the fluorescence signal observed in the second compound leaf in a longitudinal section at 49 dpi, scale bars = 100 um; (G) no fluorescence signal was observed in the third compound leaf in a transverse section at 49 dpi, scale bars = 100 um; and (H) no fluorescence signal was observed in the third compound leaf in a longitudinal section at 49 dpi, scale bars = 250 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
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Figure 4. Photographs showing the symptoms of alfalfa plants infected with Fusarium acuminatum at 49 days post-inoculation (dpi). (A) A healthy plant mock-inoculated with sterile water at 49 dpi; (B) a plant inoculated with F. acuminatum showing obvious withering and wilting branches at 49 dpi.
Figure 4. Photographs showing the symptoms of alfalfa plants infected with Fusarium acuminatum at 49 days post-inoculation (dpi). (A) A healthy plant mock-inoculated with sterile water at 49 dpi; (B) a plant inoculated with F. acuminatum showing obvious withering and wilting branches at 49 dpi.
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Figure 5. The colonization of Fusarium acuminatum in the immature pods of alfalfa at 14 days post-inoculation (dpi). (A) A hygromycin-resistant F. acuminatum mutant strain, HM29-05, expressing green fluorescent protein (GFP) sprayed on immature spiral pods of the alfalfa plant var. ‘Zhongmu NO.1′ showing the colonization of the pod skin tissue at 14 dpi, scale bars = 50 um; (B) a few hyphae extended to the central vascular bundle of pod skin tissue at 14 dpi, scale bars = 75 um; (C), the mycelium colonized the seed coat tissue at 14 dpi, scale bars = 25 um; (D) no fluorescence signal in the cotyledon tissue, scale bars = 250 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
Figure 5. The colonization of Fusarium acuminatum in the immature pods of alfalfa at 14 days post-inoculation (dpi). (A) A hygromycin-resistant F. acuminatum mutant strain, HM29-05, expressing green fluorescent protein (GFP) sprayed on immature spiral pods of the alfalfa plant var. ‘Zhongmu NO.1′ showing the colonization of the pod skin tissue at 14 dpi, scale bars = 50 um; (B) a few hyphae extended to the central vascular bundle of pod skin tissue at 14 dpi, scale bars = 75 um; (C), the mycelium colonized the seed coat tissue at 14 dpi, scale bars = 25 um; (D) no fluorescence signal in the cotyledon tissue, scale bars = 250 um. All images were captured using laser scanning microscopy (LSM); the LSM settings for GFP were 488 nm for excitation and 543 nm for emission.
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Figure 6. Photograph showing morphological characteristics of re-isolated strains from the root, stem, junction between root and stem, and fresh pods of alfalfa plants. (A) The front colony morphology of the re-isolated strain; (B) the back colony morphology of the re-isolated strain; (C) the conidia were excited to a strong green fluorescence under the fluorescence microscope, scale bars = 50 um; (D) the hyphae were excited to a strong green fluorescence under the fluorescence microscope, scale bars = 75 um.
Figure 6. Photograph showing morphological characteristics of re-isolated strains from the root, stem, junction between root and stem, and fresh pods of alfalfa plants. (A) The front colony morphology of the re-isolated strain; (B) the back colony morphology of the re-isolated strain; (C) the conidia were excited to a strong green fluorescence under the fluorescence microscope, scale bars = 50 um; (D) the hyphae were excited to a strong green fluorescence under the fluorescence microscope, scale bars = 75 um.
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Figure 7. Infection pattern of Fusarium acuminatum in alfalfa.
Figure 7. Infection pattern of Fusarium acuminatum in alfalfa.
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Wang, L.; Yang, J.; Jia, R.; Chen, Z.; Wang, N.; Wu, J.; Chen, F.; Zhang, Y.; Lin, K. Infection Process of Alfalfa Root Rot Caused by Fusarium acuminatum. Agronomy 2024, 14, 2157. https://doi.org/10.3390/agronomy14092157

AMA Style

Wang L, Yang J, Jia R, Chen Z, Wang N, Wu J, Chen F, Zhang Y, Lin K. Infection Process of Alfalfa Root Rot Caused by Fusarium acuminatum. Agronomy. 2024; 14(9):2157. https://doi.org/10.3390/agronomy14092157

Chicago/Turabian Style

Wang, Le, Jianfeng Yang, Ruifang Jia, Zhengqiang Chen, Na Wang, Jie Wu, Fangqi Chen, Yuanyuan Zhang, and Kejian Lin. 2024. "Infection Process of Alfalfa Root Rot Caused by Fusarium acuminatum" Agronomy 14, no. 9: 2157. https://doi.org/10.3390/agronomy14092157

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