Detection and Characterization of Lasiodiplodia pseudotheobromae Associated with Stem Wilt on Ficus hirta (Vahl) and Its Fungicidal Sensitivity

Abstract

Ficus hirta Vahl is an important medicinal and edible plant in southern China. Typical green wilting on leaves and brown necrotic spots on the stems were observed since mid-June 2022 in an F. hirta plantation in Danzhou, Hainan (China). The disease rapidly developed, causing stem withering and plant death. The disease incidence varied from 45 to 85% prevalence, and the average disease index was 47 in the period of outbreak during June to December. Relevant hypothetical fungi were isolated from naturally infected wilt tissues, and their pathogens were preliminarily confirmed to be Lasiodiplodia pseudotheobromae through hypothetical fungal culturing, morphological characteristic observations, and pathogenicity testing on F. hirta plants. The phylogenetic tree constructed based on partial ITS, translation elongation factor (TEF1-α), and the β-tubulin gene (TUB2) further confirmed the identity of the pathogen as L. pseudotheobromae. Further research on the biological characteristics of L. pseudotheobromae showed that the optimal temperature for the growth of L. pseudotheobromae was PDA medium, with a temperature of 30 °C and pH of 6. Peptone and fructose were the optimal nitrogen and carbon sources for it. In vitro efficacy testing showed that among eleven fungicides, fluazinam and prochloraz had the highest mycelial growth inhibition, with an EC₅₀ of 0.0477 µg/mL and 0.0996 µg/mL, respectively. And the two fungicides showed significant control on the stem wilt of F. hirta in a pot. To our knowledge, this is the first comprehensive report on the pathogen identification and biological characteristics of L. pseudotheobromae infecting the stem wilt of F. hirta in China. Our results provide important information for developing effective management measures and controlling this disease.

1. Introduction

Ficus hirta Vahl (family: Moraceae) has great edible and medicinal value. In China, F. hirta is mainly distributed in the tropical and subtropical regions of mountains, forest edges, shrubs, and sparse forests, at altitudes of 500–1000 m in Guangdong, Guangxi, Hainan, Fujian, Guizhou, Yunnan, and Jiangxi. The roots of F. hirta are cooked with other foods, giving the food a special rich coconut flavor, and often used as a folk medicinal diet ingredient, having the “Guangdong ginseng” reputation [1,2] (pp. 160–162, 194). F. hirta is rich in protein, polysaccharides, and flavonoids. Traditionally, the roots are used for treating spleen deficiency edema, weakness, consumption cough, rheumatism and arthralgia, and other diseases, with a remarkable curative effect [3] (p. 29). Moreover, related studies have also revealed that F. hirta has the distinct ability to eliminate cough, sputum, and stridor [4]; enhance immunity [5]; and exhibit protective effects on liver injury [6], and it has anticancer properties [4] and beneficial preventive and therapeutic effects on non-alcoholic fatty liver disease [7]. Awareness of its multiple medicinal and nutritional values in food has increased the demand for F. hirta or products containing F. hirta, leading to a shortage of supply of this species. In China, the organized cultivation of F. hirta mainly takes place in Guangdong, Guangxi, and Hainan provinces. However, with the increase in planting area, new diseases constantly appear in F. hirta, some of which affect commercial cultivation area and crop yield. At present, there are almost no reports of disease studies focusing on F. hirta in China.

In a June 2022 survey, symptoms of stem wilt disease with brown lesions on the stem were observed in the experimental field of F. hirta at CATAS (experimental farm team three), Danzhou, Hainan. Within a few days of infection, the whole leaves and stems of the crop were found to be faded and withered. The occurrence of stem wilt in F. hirta revealed a large-scale infection. The disease incidence was found to vary from 45 to 85% prevalence, and the average disease index was 47 in the period of outbreak of June to December. In fields severely affected by the disease, all plants withered and died, causing significant economic losses. Based on the rapid spread of the disease and the symptoms of death on other plant areas (branches), it was determined that it may be caused by infection by fungi of the Botryosphaeriaceae. The Botryosphaeriaceae are common dieback and canker pathogens of woody host plants, including some fruit trees [8]. Lasiodiplodia species are members of the Botryosphaeriaceae. Among these, L. pseudotheobromae was segregated from L. theobromae [9]. L. pseudotheobromae has a wide host range and is an important class of plant endophytic and pathogenic fungi, acting as a pathogen causing or associated with canker, wilt, dieback, collar rot, or fruit rots [10,11]. The occurrence of these diseases seriously affects the products and quality of crops. As the disease was previously unknown, the present study was undertaken to investigate the causal agent of stem wilt in F. hirta based on morphological characteristics, supplemented with molecular characterization. Further, chemical fungicides were evaluated for their antifungal activity in indoor and pot experiments, with the results to be used in devising management strategies for stem wilt disease in F. hirta.

2. Materials and Methods

2.1. Sample Collection and Fungal Isolation

Experiments were conducted during June 2022 by experimental farm team three at the CATAS, Danzhou, Hainan, China, located at 19°34′ N and 109°29′ E. The climate of the experimental site is tropical monsoon. The main soil type is latosol yellow soil. In dry land, the main crop is intercropping rubber tree. Before the experiment, the soil had a pH of 4.34, with 16.19 g kg⁻¹ organic matter, 0.11 g kg⁻¹ hydrolysable N, 1.56 mg kg⁻¹ rapidly available P, and 27.06 mg kg⁻¹ rapidly available K in the 0–10 cm soil layer. Stem samples showing disease symptoms were collected separately in a sealed plastic bag from the experimental field of the CATAS experimental farm, Danzhou, Hainan, and brought to the laboratory of CATAS Environment and Plant Protection Institute, Haikou, Hainan, for further study.

To isolate the associated pathogen, symptomatic stem samples were isolated and rinsed under tap water to remove attached soil debris and dust. The visible infected lesions on stems were cut into 3 mm × 3 mm pieces and surface-sterilized with 75% alcohol for 1 min followed by 2% sodium hypochlorite (NaClO) for 2 min, then washed three times with sterile distilled water. Excess water from the samples was removed by soaking on sterile filter papers and blot-drying. The sample pieces were then transferred onto potato dextrose agar (PDA) medium containing 100 μg ml⁻¹ ampicillin. These inoculated plates were kept at 28 °C in an incubator in the dark for 3–5 days [12,13].

2.2. Pure Culture and Morphological Identification

The mycelium growth was observed after 72 h of incubation and pure culture of the fungus was retrieved on PDA from single spore isolation following the standard procedures [14]. Recorded observation of growth and other morphological parameters from pure culture.

2.3. Koch’s Postulate Test

To verify the pathogenicity of stem wilt disease causal agent in F. hirta, an evaluation was conducted on healthy 4 month-old F. hirta potted seedlings (stem diameter 7–9 mm). These healthy seedlings were tissue-cultured by researchers from CATAS—Rubber Research Institute. The surfaces of healthy stems were disinfected with 75% ethanol, and then the stems of F. hirta were pricked with a sterilization needle and inoculated on two mycelium disks (5 mm diameter) cultured at 28 °C for 2 days, inoculated with sterilized PDA agar block as a control, with three seedlings for each treatment. The inoculated seedlings were kept in a natural condition at 25 °C–30 °C with moistened sterile cotton to preserve humidity and bound with plastic wrap. Observations of symptom development were carefully recorded at regular constant intervals. The Fungus was re-isolated from the inoculated diseased stems, and their morphological and cultural characteristics were compared with pathogens previously isolated from diseased F. hirta stems.

2.4. DNA Extraction, PCR Amplification and Phylogenetic Analysis

Further identification of the isolated pathogens was carried out by partial sequencing of internal transcribed spacer (ITS), translation elongation factor-1α (TEF1-α, and β-tubulin (TUB2) gene. Pure cultured fungus was inoculated onto PDA medium and incubated at 28 °C for 2 days for multiplication. The mycelium was harvested and finely ground into powder in liquid nitrogen. The manufacturer’s instructions for extracting the fungal genomic DNA were followed using the fungal DNA Kit (Omega Bio-tek, Inc., Beijing, China). Using the extracted DNA as a template, primers were employed for multi-locus gene amplification and sequencing. The ITS1 and ITS4 primers were utilized to amplify the internal transcribed spacer region from ITS1-5.8S to ITS4 in fungi [15] (pp. 315–322). Furthermore, to precisely identify the species and molecular traits of the relevant Lasiodiplodia sp., specific primers were utilized for PCR amplification of parts of the translation elongation factor (TEF-1α) gene [9] and β-tubulin gene (TUB2) [16].

The PCR reaction was carried in a total volume of 25 μL, comprising 2 μL of template DNA, 12.5 μL 2 × E-Taq pre-Mix, 1.0 μL of forward and reverse primers each 10 μmol/mL, and sterile nuclease free water to compensate the volume. It began with one cycle at 95 °C for 3 min, followed by 33 cycles of 95 °C for 30 s, annealing for 30 s (ITS: 56 °C; TEF-1α: 55 °C; TUB2: 59 °C), and 72 °C for 45 s, concluding with a final extension at 72 °C for 10 min prior to PCR measurement. The amplified products were assessed by gel electrophoresis (1.5% w/v) on an agarose gel. The amplified products were subsequently recovered and subjected to bidirectional sequencing by Beijing Liuhe Huada Gene Technology Co., Ltd. (Beijing, China).

The retrieved sequences were primarily processed by BLASTN analysis of NCBI (Table S1) for the presumptive identification of the pathogen. The homologue nucleotide sequences for the ITS, TEF-1α, and TUB2 regions were sourced from the NCBI database and underwent multiple alignments using the Clustal W algorithm of BioEdit 7.1.3. Based on the phylogenetic analysis using the maximum likelihood tree phylogeny of MEGA11.0 software, the neighbor-joining (NJ) approach [17] was employed to construct phylogenetic trees from three genes sequences, and 1000 bootstrap replicates were conducted [18].

2.5. Determination of Biological Characteristics of the Pathogen

The fungi mycelia used in this study were all of 5 mm-diameter agar plugs cut from the edges of the pathogen cultures grown on PDA medium for 2 days, and inoculated at the center of the plates’ medium, and each treatment in this study was replicated thrice. To clarify the biological of characteristics of the pathogen, the mycelial plugs were inoculated onto PDA, OMA, CMA, CA, Czapek, PCA, WA, NA, tomato juice agar medium (TJA), and F. hirta juice agar medium (FJA) (Table S2) and cultured at 28 °C for 2 days; ten different temperatures of 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, 32 °C, 35 °C and 37 °C; and eleven different pH levels of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0. In order to clarify the effects of carbon and nitrogen sources on the growth of the pathogen, Czapek media were produced using equal amounts of glucose, sucrose, fructose, maltose, sorbitol, mannitol, D-xylose, alpha lactose, soluble starch, and glycerol as the sole carbon sources. Equal amounts of beef extract, yeast extract powder, sodium nitrate, ammonium nitrate, urea, glycine, L-cysteine, ammonium chloride, ammonium sulfate, peptone, and potassium nitrate were used from different nitrogen sources as quality nitrogen sources for Czapek culture medium. Moreover, Czapek medium, without carbon or nitrogen sources as a control, was used to inoculate a pathogen of 5 mm-diameter agar plugs, which was cultured at 28 °C for 2 days. The colonies’ diameters were measured using the cross method.

2.6. Evaluation of Fungicide Sensitivity

Eleven commercial chemical fungicides were obtained from CATAS-Environment and Plant Protection Institute, Haikou, Hainan province (

Table 1. Effective constituent and series concentration of the tested fungicides.

Fungicides	Manufacturer	Concentration (µg/mL)
Fluazinam (98% a.i.)	Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China	0.4, 0.2, 0.1, 0.05, 0.025
Thiophanate-Methyl (95% a.i.)	Anlin Biochemical Co., Ltd., An yang, China	10, 5, 2.5, 1.25, 0.625
Difenoconazole (95% a.i.)	Beijing Green Nonghua Plant Protection Technology Co., Ltd., Beijing, China	2, 1, 0.5, 0.25, 0.125
Prochloraz (97% a.i.)	Jiangsu Huifeng Biological Agriculture Co., Ltd., Yancheng, China	0.5, 0.25, 0.125, 0.063, 0.031
Propiconazole (95% a.i.)	Shanghai Demohua Agricultural Chemical Co., Ltd., Shanghai, China	1, 0.5, 0.25, 0.125, 0.063
Tebuconazole (96% a.i.)	Jiangsu Fengdeng Crop Protection Co., Ltd., Changzhou, China	0.4, 0.2, 0.1, 0.05, 0.025
Chlorothalonil (98% a.i.)	Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China	50, 10, 5, 1, 0.5
Carbendazim (97% a.i.)	Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China	5, 1, 0.5, 0.1, 0.05
Iprodione (97% a.i.)	Hubei Wanye Pharmaceutical Co., Ltd., Wuhan, China	1, 0.5, 0.25, 0.125, 0.063
Azoxystrobin (95% a.i.)	Shanghai Demohuaxue Technology Co., Ltd., Shanghai, China	40, 20, 10, 5, 2.5
Mancozeb (96% a.i.)	Shanghai Demohuaxue Technology Co., Ltd., Shanghai, China	20, 10, 5, 2.5, 1.25

), and their effectiveness against fungi was evaluated under in vitro conditions. The experiments were carried out to measure the fungicides’ inhibition effect using the mycelia growth rate method. Five different concentrations of the fungicides were separately mixed into sterilized PDA, and amended PDA was poured into culture dishes and allowed to solidify. The solidified PDA plates were inoculated with a 5 mm mycelium disk cut from 2-day-old pure fungal cultures. Each treatment was repeated three times, and the growth of fungal colonies was measured 2 days after inoculation. After subtracting 5 mm (mycelium disk measurement) from the recorded growth, the radial growth was analyzed. The inhibition percentage of the mycelium growth relative to the control group was calculated using the following formula [19]: using the probability of conversion of inhibition rate as the longitudinal coordinate (y), and the logarithm of concentration as the transverse coordinate (x), we derived the regression equation y = ax + b, the median inhibitory concentration (EC₅₀), and the correlation coefficient (R) [20]. Two fungicides with low EC₅₀ value were selected for the pot experiment.

$$\mathrm{PI}=\left(C-T\right)\times 100/C$$

(1)

where

PI = Inhibition percent;

C = Colony diameter of the fungus in absent of treatment;

T = Colony diameter of the fungus with treatment.

The pot experiments of stem wilt of F. hirta were conducted during 2023 in augmented randomized block design (RBD). The seedlings of F. hirta cultivated for 4 months were planted under natural conditions with controlled water and fertilizer application. A total of seven treatments were applied, including one control (Check), with three repeats for each treatment. For each treatment of nine seedlings, we gently pricked the stem of F. hirta with a sterilization needle and inoculated two mycelial disks, moisturized them with sterile cotton, and then bound them with plastic wrap. We sprayed the fungicide 12 h after inoculating the mycelium disks, and sprayed again 3 days later. In the experimental plot, the observation results of the disease index were recorded 7 days after spraying. The disease index and control effect of stem wilt in F. hirta were calculated according to standard method. See

Table 2. Stem disease classification criteria.

Grade	Value	Degree of Disease Occurrence
Level 0	0	The main stem has no diseased spots
Level 1	1	The length of the main stem lesion around the stem accounts for less than 25% of the circumference of the main stem
Level 2	2	The length of diseased spots around the main stem accounts for 25% to 50% of the circumference of the main stem
Level 3	3	The length of diseased spots around the main stem accounts for 50% to 75% of the circumference of the main stem
Level 4	4	The length of the main stem lesion around the stem accounts for more than 75% of the circumference of the main stem, or the stem withers and dies

for disease grading standards [21].

$$\mathrm{DI}={\sum }_i\left(A_i\times B_i\right)\times {\displaystyle \frac{100}{\left(4\times C\right)}}$$

(2)

where

DI = Disease index;

i = The severity level of the disease is 0, 1, 3, 5, 7, and 9, in order;

A = Number of infected lesions at each level;

B = Relative level value;

C = Total number of infected lesions observed.

$$\mathrm{PE}=\left(C-T\right)\times 100/C$$

(3)

where

PE = Control effect;

C = Disease index with absence of treatment area;

T = Disease index with treatment area.

2.7. Statistical Analysis

Data were subjected to analysis by Duncan’s statistical test of variance, appropriate for the RBD, by using the Microsoft Excel package, version 2016 (USA). The results were presented at 5% level of significance (p = 0.05). The critical difference (CD) values were calculated to compare the various treatment means.

3. Results

3.1. Disease Symptoms and Morphology of Pathogen

The symptoms of stem wilt include leaf dehydration, green wilting, and brown necrotic spots on the stems, which rapidly develop upwards and downwards. Necrotic spots gradually developed, with dark brown edges merging and covering most of the stem, which gave a withered appearance and eventually dried (Figure 1).

A total of ten strains with the same colon morphology were isolated from the withered stem sample by tissue separation method, and named MA1–MA10. The representative strain MA2 with good growth was selected for morphological observation. The pure culture of isolated fungus produced radially white, round or irregular colonies, which turned grayish white after some time and had an abundance of aerial mycelia in culture (Figure 2a–c). The average colony diameter was 85.50 mm on the PDA plates after 29 h incubation. The conidia were single-celled, colorless, transparent, nearly oval or oval, later becoming double cells and becoming dark brown, with a septum in the center, a transparent cylindrical structure and measuring (13.36–23.51) μm × (10.48–12.65) μm (n = 50) (Figure 2d–f). Therefore, based on its morphological characteristics, the fungus was identified as Lasiodiplodia pseudotheobromae species.

3.2. Pathogenicity Test

Two days after inoculation with MA2 mycelial blocks, the inoculated stem of F. hirta showed the typical stem wilt symptom of L. pseudotheobromae as observed in the field, while no disease symptoms were observed on the control plants (Figure 3a). When the stems were pricked and inoculated with MA2 for 2 days, the leaves turned yellow, wrinkled, or even fell off, and brown spots and white mycelium adhered to the stem (Figure 3b). Large areas of brown necrotic spots spread up the stem on the fourth day of inoculation (Figure 3c); the disease spot further expanded and formed a dark brown lesion, with the stem wilting and the presence of black pycnidial arising on the 6th day of inoculation (Figure 3d). The same fungi were re-isolated from an artificially inoculated disease stem, thereby conforming to Koch’s postulates.

3.3. Sequence, Identification of Pathogen Species and Phylogenetic Analysis

The amplicon of 513 bp, 532 bp and 444 bp size was retrieved using ITS, TEF-1α and TUB2 primers, purified and sequenced through outsourcing, respectively. The assembled sequences of the partial ITS, TEF-1α and TUB2 regions were submitted to NCBI GenBank with accession numbers OP897010.1, OQ053274.1 and OP918017.1, respectively. The sequence of the ITS region of MA2 isolated from F. hirta showed 100% similarity in the BLASTN similarity search with isolates of L. pseudotheobromae (MT332314, MN341226, MN341225, etc.).

Further, combined analysis of all three, ITS, TEF-1α and TUB2, gene sequences (Figure 4) found them to consist of 14 taxa, which comprised L. pseudotheobromae isolate MA2 obtained in this study and 13 additional isolates including the outgroups Botryosphaeria stevensii and Botryosphaeria obtusa. According to the multi-gene combined phylogenetic tree displayed, isolate MA2 was clustered with L. pseudotheobromae isolate, where CBS116459 is the same branch, and the node support rate is 100%. Combined with morphological characteristics, MA2 isolated from diseased stems of F. hirta plants in Hainan province, China, was identified as L. pseudotheobromae.

3.4. Biological Characterization of Lasiodiplodia pseudotheobromae

The strain MA2 could grow on PDA, OMA, CMA, CA, Czapek, PCA, WA, NA, TJA, and FJA media (Figure 5A). The best mycelium growth rate was found on PDA, with an average colony diameter of 69.00 mm after 29 h of cultivation; next was on NA, with an average colony diameter of 57.00 mm. The mycelial growth was most compact on PDA and NA. Among these, the mycelial growth rate on WA medium was the slowest, with an average colony diameter of only 24.25 mm. Therefore, PDA was considered to be the most suitable medium for the growth of L. pseudotheobromae, and it was used for the determination of the optimal growth conditions for MA2 in lateral experiments. When fructose was used as the carbon source, the utilization rate was the highest, the colony growth rate was significantly higher than other carbon source media, and the average colony diameter was 59.25 mm; the utilization efficiency of the strain for α-lactose, glucose, and sucrose was second, while the utilization efficiency for mannitol and D-xylose was slightly lower, and the average colony diameter was less than 42.00 mm (Figure 5B). Additionally, the strain had the highest utilization rate when using peptone and yeast extract powder as nitrogen sources, the mycelium densities were the highest and the growth rates were the fastest, and the average colony diameter was about 57.00 mm; next were beef extract and ammonium nitrate; when urea was used as the nitrogen source, colony diameter grew at the slowest rate, with the average colony diameter of only 2.75 mm, which was significantly lower than the utilization rate of other nitrogen sources (Figure 5C). Strain MA2 could grow within the temperature range of 10 °C to 37 °C (Figure 5D). When the temperature was below 5 °C, the mycelium stopped growing. Within the temperature range of 5 °C to 10 °C, the growth of the mycelium was inhibited, with a maximum average diameter of the colony of only 1.00 mm; within the range of 10 °C to 30 °C, the growth rate of mycelium accelerated with the increase in temperature. At 30 °C, the mycelial growth rate was fastest, with an average colony diameter of 77.25 mm, which was the optimal growth temperature for this pathogen. Within the temperature range of 30 °C to 37 °C, mycelial growth was inhibited and the growth rate slowed down significantly. The optimal pH for mycelial growth was 6, and the average diameter of the colony was 68.50 mm. The pathogen grew rapidly with the pH range of 4.0 to 7.0. When the pH was below 3.0 or above 8.0, mycelial growth was inhibited (Figure 5E).

3.5. Evaluation of Fungicide Sensitivity

The results in

Table 3. In vitro efficacy of fungicides against Lasiodiplodia pseudotheobromae.

Fungicides	Regression Equation	Correlation Coefficient (r)	EC50 (µg/mL)
Fluazinam	y = 1.4675x + 6.9392	0.9803	0.0477
Prochloraz	y = 0.8410x + 5.8425	0.9967	0.0996
Tebuconazole	y = 1.1757x + 6.1145	0.9941	0.1127
Iprodione	y = 3.7339x + 8.4946	0.9950	0.1159
Propiconazole	y = 1.0861x + 5.9834	0.9895	0.1243
Carbendazim	y = 1.5813x + 5.8423	0.9856	0.2933
Difenoconazole	y = 1.5047x + 5.6738	0.9945	0.3566
Thiophanate-Methyl	y = 2.5114x + 4.7267	0.9993	1.2847
Mancozeb	y = 1.6801x + 4.7240	0.9753	1.4598
Chlorothalonil	y = 1.1061x + 4.7049	0.9559	1.8485
Azoxystrobin	y = 1.3411x + 3.8461	0.9934	7.2519

show that compared with the control treatment, the eleven evaluated fungicides significantly inhibited the mycelium growth of the test fungus L. pseudotheobromae under in vitro conditions. Among the tested fungicides, fluazinam 98% and prochloraz 97% were found most efficient inhibition the pathogen of mycelial growth, with EC₅₀ values of 0.0477 µg/mL and 0.0996 µg/mL, respectively, followed by Tebuconazole 96%, Iprodione 97%, Propiconazole 95%, Carbendazim 97% and Difenoconazole 95%, with EC₅₀ values of 0.1127 µg/mL, 0.1159 µg/mL, 0.1243 µg/mL, 0.2933 µg/mL and 0.3566 µg/mL, respectively. Azoxystrobin 94% was found to be least effective, with an EC₅₀ of 7.2519 µg/mL mycelium growth inhibition over the control PDA mycelium growth.

Additionally, two fungicides were evaluated against L. pseudotheobromae, showing significant control on the stem wilt of F. hirta (

Table 4. Control effect of two fungicides on the stem wilt disease of Ficus hirta.

Fungicides	Manufacturer	Concentration (µg/mL)	7 Days after Second Use
			Disease Index	Control Effect * (%)
Prochloraz (25% EC)	Andao Maihuifeng (Jiangsu) Co., Ltd., Yancheng, China	167	30.56	47.45a
		250	31.95	45.06a
		500	27.78	52.55a
fluazinam (50% SC)	Shiyuan (Shanghai) Chemical Co., Ltd., Shanghai, China	222	38.89	32.43a
		333	31.94	45.04a
		667	36.11	37.37a
CK	/	0.00	58.33	0.00

* Lowercase letters indicate a statistical significance level of 0.05, and there is no significant difference between levels using the same lowercase letters.

). The control effects of prochloraz 25% and fluazinam 50% were 45.06~52.55% and 37.37~45.04%, respectively.

4. Discussion

Ficus hirta is one of the important medicinal and edible plants grown in the mountain and forest areas of southern China. The crop roots harvested from mountain areas, forest, and cultivation areas are supplied to pharmaceutical companies for various formulations. However, a new disease was observed in the experimental field of F. hirta at CATAS-Experimental Farm Team Three in 2022, and occurred at both seedling and adult stages, with leaves and stems of F. hirta drying up during the rainy season. Diseases spreads rapidly in F. hirta, responsible for the destruction of crop production. In this study, the crops infected with stem wilt disease and the related pathogenic fungus was identified as Lasiodiplodia pseudotheobromae based on morphology and molecular sequences. L. pseudotheobromae is an important plant pathogen with a wide range of hosts, which can infect the branches and fruits of various plants, causing stem canker [22,23], stem rot [24], stem blight [25], leaf wilt [26], fruit rot [27] and other symptoms, which bring huge economic losses to agricultural production [9,28,29,30,31]. Because the morphologies of L. pseudotheobromae and L. theobromae are highly similar, L. pseudotheobromae has been described as the hidden species in the L. theobromae complex, but there are obvious differences in conidial morphology between the two species. In addition, the clustering on the phylogenetic tree was different. In 2008, L. pseudotheobromae was divided into a new species [9], which, as previously reported, can infect rubber trees and cause leaf spots [32] and branches to die [33]. The branch blight disease of blueberry and apple first reported in China was caused by L. pseudotheobromae [34,35]. At present, there are few stem diseases caused by L. pseudotheobromae, and there are no reports of infection in medicinal plants by L. pseudotheobromae. Therefore, in this study, in addition to the morphological keys, advanced molecular tools (ITS, TEF-1α and TUB2) were used to characterized the associated pathogens of F. hirta stem disease in order to accurately characterize Lasiodiplodia spp. Additionally, attempts were made to document symptoms, pathogenicity, biological characterization of the pathogen, and disease control strategies.

The genus Lasiodiplodia belongs to the Botryosphaeriaceae family, and most species are mainly distributed in tropical and subtropical regions [29]. Due to the similar morphological characteristics among Lasiodiplodia species, it is impossible to achieve a clear identification of pathogen species by using traditional morphological features [9]. The morphological structure of L. pseudotheobromae is similar to L. theobromae in this genus [36] (pp. 1–2); the two species are not easily distinguishable, but they differ in the morphology of their conidia. The mature conidia of L. pseudotheobromae are larger and ellipsoidal in shape, rather than tapered to the base and conical like L. theobromae. In addition, according to molecular identification and phylogenetic analysis, L. pseudotheobromae and L. theobromae can be clearly distinguished, and the two species are clustered in different branches of the phylogenetic tree. Therefore, the identification of Lasiodiplodia species should be combined with morphology and molecular biology [34].

The pathogenicity of isolate (MA2) and identification of the pathogen was determined based on morphological features, which is a prerequisite and indispensable part of separating fungal species [37]. In addition to the morphological characterization of L. pseudotheobromae, the pathogen was further confirmed by ITS (OP897010.1), TEF-1α (OQ053274.1) and TUB2 gene (OP918017.1) partial sequences submitted to the NCBI database. The sequences of three genes shared >99% identity in a BLASTN search of GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 29 May 2023) with L. pseudotheobromae isolates. In several previous studies, ITS sequences have been used to identify L. pseudotheobromae from different hosts [32,34,35]. Besides ITS sequencing, the wilt-disease-associated Lasiodiplodia sp. was characterized based on the sequencing of partial TEF-1α and TUB2 genes, which is essentially required for the accurate identification of Lasiodiplodia species [11,32,34,35]. A phylogenetic tree was constructed, and the analysis further confirmed L. pseudotheobromae. The pathogenicity of the characterized isolate MA2 was established by inoculating on the healthy plants, and it produced an identical symptom to that observed in the naturally infected plants.

Exploring the biological characteristics of pathogens helps to understand the impact of environmental conditions on disease occurrence and transmission, and provided a theoretical basis for disease prevention and control. In this study, L. pseudotheobromae isolated from the diseased stems of F. hirta grew fastest at 30 °C and pH 6.0, and PDA was the optimal medium for the growth of the strain. The utilization rates of fructose, yeast extract powder, and peptone were the highest. The strain stopped growing below 5 °C, slowed down significantly at 30 °C to 37 °C, and grew better under acidic conditions than alkaline conditions, with an average colony diameter at its largest at a pH of 4 to 7. The optimal biological characteristics for the growth of MA2 in this study were similar to those of the L. pseudotheobromae causing leaf spot disease in rubber trees [32], and the strain causing leaf spot optimal growth temperature was of 32 °C and pH 5; the highest utilization rates were with D-fructose and yeast extract. It can be seen that the mycelium of L. pseudotheobromae grows well under high temperatures, and an environment of high temperature and high humidity is conducive to promoting the spread of the disease. Hainan province belongs to a tropical monsoon climate, and the occurrence of this disease is consistent with the fact that the stem wilt on F. hirta mainly occurs in summer and the rainy season.

Chemical control has the characteristics of high efficiency and reliability, and occupies an irreplaceable position in crop disease management. In order to develop control strategies, eleven fungicides were evaluated against the L. pseudotheobromae. Fluazinam 98% and prochloraz 97% were found to be most efficient in the inhibition of mycelial fungal growth under in vitro conditions, with EC₅₀ values of 0.0477 µg/mL and 0.0996 µ g/mL, respectively. The order of the inhibitory effects of the 11 tested fungicides on L. pseudotheobromae is as follows: fluazinam > prochloraz > tebuconazole > iprodione > propiconazole > carbendazim > difenoconazole > thiophanate-methyl > mancozeb > chlorothalonil > azoxystrobin. Similar results were observed in other studies (in vitro) against L. pseudotheobromae using fluazinam and prochloraz [38]. In addition, the two fungicides had obvious control effect on the stem wilt of F. hirta in pot experiments. Prochloraz belongs to the imidazole fungicides, which can inhibit the biosynthesis of ergosterol in pathogenic fungi, disrupt the structure of fungal cell membranes, and inhibit and kill fungi [39]. Fluazinam belongs to the pyridine fungicides and is a new type of broad-spectrum fungicide that replaces aniline, with no cross resistance with benzimidazole, dicarboximide, and existing fungicides on the market [40]. The chemical fungicides screened in this study have a decreasing effect on the potted control of stem wilt in F. hirta compared to other scholars’ research results. On the one hand, this may be due to differences in the types and prevalence of the disease, as well as the impact of field application of fungicides; on the other hand, this may be due to L. pseudotheobromae having a fast growth rate (growing to 9 cm after 29 h of cultivation in PDA medium) and strong infectivity, and it being difficult to rapidly inhibit the spread of pathogens by spraying chemical fungicides, resulting in differences in control effects. Therefore, the sensitivity of its fungicide needs to be further confirmed in the field later based on the first discovery of leaf spot disease on rubber trees caused by L. pseudotheobromae infection in a rubber plantation in Sanya City, Hainan Province of China [32]. Due to the potential harm of this disease to rubber trees and other crops, it should be given great attention in production, and monitoring, prevention, and control should be strengthened.

5. Conclusions

In summary, ten fungal strains were isolated from diseased stems of Ficus hirta plants, and a new pathogen of F. hirta stem wilt was identified as Lasiodiplodia pseudotheobromae. The biological characteristics of L. pseudotheobromae were clarified, and toxicity tests were conducted on 11 fungicides to screen for prochloraz and fluazinam, which have strong toxicity to pathogens. Potted plant experiments have shown that these two selected fungicides can effectively control stem blight of F. hirta. Our research results can provide a foundation for a deeper understanding of the etiology, prevalence, and effective control strategies of stem wilt in F. hirta.