The polyphagous pathogen Lasiodiplodia theobromae (Pat.) Griffon and Maubl. (1909) belongs to the family Botryosphaeriaceae (Dissanayake et al., 2016; Zhang et al., 2021) and causes multiple disease symptoms on approx. 500 plants (Punithalingam, 1976). In Peru, this pathogen has been described as a causal agent of dieback and wood necrosis in blueberry (Rodríguez-Gálvez et al., 2020), mango (Rodríguez-Gálvez et al., 2017), avocado (Rodríguez-Gálvez et al., 2021) and table grapes (Rodríguez-Gálvez et al., 2015). As symptom severity and yield losses strongly depend on the virulence of isolates (Gunamalai et al. 2023), a reliable method for determining their aggressiveness is indispensable for performing efficient disease control measures. Virulence tests with L. theobromae isolates have been carried out using two methods, i.e. inoculation into the middle part of the stem after wounding (Bautista-Cruz et al., 2019; Berraf-Tebbal et al., 2020; Dong et al., 2020; Hernández et al., 2023; Úrbez-Torres et al., 2008; Úrbez-Torres et al., 2009; El-Ganainy et al., 2022; Coutinho et al., 2017; Rangel-Montoya et al., 2021; Briste et al., 2022; Kong et al., 2023), or onto excised apical bud of the stem (Alama et al., 2006; Billones-Baaijens et al., 2013; Hernández et al., 2023; Kwon et al., 2017; Polashock and Kramer, 2006; Rodríguez-Gálvez et al., 2015; Rodríguez-Gálvez et al., 2017; Rodríguez-Gálvez et al., 2020 and Rodríguez-Gálvez et al., 2021). In spite of the fact that distinct inoculation methods have been employed, rigorous comparisons of these methods have not been carried out. The present study was performed to determine which of the two inoculation methods yields more severe dieback and wood necroses symptoms and is therefore more suitable to determine virulence of isolates.
In this study, 10 months old avocado plants (Persea americana cv. Hass) plants grafted on West Indian rootstock were inoculated with the virulent L. theobromae isolate LA-VLCA3 obtained from avocado branches with dieback symptoms (Rodríguez-Gálvez et al., 2021). This isolate was plated onto 2% (w/v) potato dextrose agar (PDA) (HiMedia Laboratories Pvt.Ltd., Dindhori, Nashik, India) and incubated at 30°C for 72 h.
Plants were inoculated using L. theobromae-covered agar blocks taken from the edge of a colony. This inoculation method allowed comparison of the data with those obtained in previous experiments (e.g. Rodríguez-Gálvez, et al 2015, 2017, 2020, 2021; Úrbez-Torres and Gubler, 2009; Úrbez-Torres at al. 2008). Moreover, we decided to use agar block inoculation because inoculation of conidial suspensions into wounds in vertical stems may result in major loss of conidia due to the outflow of an unknown volume of the inoculum. Mid-stem inoculation was done after wounding with a sterile corkborer (5 mm diameter) immediately above the grafting zone. An agar disc with mycelium (4 mm diameter) of the pathogen obtained from the edge of a colony at 72 hours post inoculation (hpi) was deposited into the wound and the inoculated area was covered with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (Úrbez-Torres et al., 2008). For apex inoculation, the apical bud was cut off with a sterile scalpel and a 4 mm agar disc with fungal mycelium (see above) was placed onto the wound. The inoculated area was covered with sterile cotton moistened with sterile distilled water and sealed with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (Rodríguez-Gálvez et al., 2015). Five plants per treatment were inoculated and incubated for 28 days in a greenhouse at an average temperature of 26°C. The experiment was repeated four times, yielding a total of 20 tested plants per treatment.
Symptom development was observed daily and the expansion of external and internal necroses caused by the pathogen was assessed at 28 days post inoculation (dpi) by measuring length of necroses from the point of inoculation to the border of the visible infection using a digital vernier. In mid stem-inoculated plants, acropetal and basipetal external and internal necroses were measured, and in plants inoculated at the excised apex, only external and internal basipetal necroses were measured.
To confirm statistical normality of necrosis progression, the SPSS-V.25 software (IBM, New York, USA) was used, applying the Shapiro-Wilk test for data smaller than 50. For comparison of means in samples that do not show normal data distribution, the non-parametric Kruskal-Wallis test was used. For comparison of means in samples from normal populations, the parametric ANOVA test was used, followed by Tukey's test (p ≤ 0.05). Statgraphics Centurion software version VII (Statgraphics Technologies Inc., The Plains, VA, USA) was employed for both comparisons.
In plants inoculated at the mid-stem, an irregular externally visible black necrotic spot was observed around the inoculation site. Developing necrosis expanded acro- and basipetally from the inoculation site (Figure 1a, arrowhead). After dissecting the cortical zone longitudinally, internal necrosis of the tissue was observed, which had a greater acropetal and basipetal extension than the externally visible necrosis (Figure 1b, arrows). Interestingly, cross-sections and longitudinal sections revealed that internal necroses first developed acro- and basipetally underneath the bark (Figure 1c, arrows), established bark-associated necroses and subsequently colonized the xylem and grew towards the pith, resulting in the sectorial necrosis typically observed in L. theobromae-infected stems (Figure 1d, arrows). Thus, development of necroses in avocado resembles those observed in L. theobromae-infected grapes (Rodríguez-Gálvez, 2021).
Following apex inoculation, necroses developed dramatically and expanded basipetally, affecting all stem tissues from the point of inoculation, and resulted in necrotizing leafs and branches at 28 dpi (Figure 2a). Formation of enormous numbers of conidia covering a large part of the infected stem was visible as whitish coating (Figures 2a and, at larger magnification, Figure 2b, co). Internal necrosis extending basipetally beyond the edge of the externally visible necrosis (Figure 2c, arrowhead) was detected after cutting the bark longitudinally (Figure 2c, arrows).
Macroscopic evaluation of infected plants (Figures 1 and 2), and quantification of the length of external and internal necroses revealed that the fungus had massively spread in the stem (internal necroses) before symptoms become externally visible (Figure 3). In mid-stem-inoculated plants, no significant differences were observed between acropetal and basipetal expansion of necroses (p = 0.432774 > ɑ=0.05), indicating similar fungal upward- and downward-directed spread. Importantly, basipetal extension of both external and internal necroses were significantly more pronounced in stems inoculated at the excised apex, as compared with mid stem-inoculated plants (p=000000006318 < ɑ=0.05) (Figure 3). Clearly, these data indicate that excised-stem-inoculation is an excellent method to evaluate virulence of field isolates of L. theobromae.
Our results show that development of necroses in stems of young avocado plants inoculated with L. theobromae is strongly affected by the inoculation method used. The advance of internal necrotization preceeded that of external necrotization, suggesting that the fungus spreads vertically within the stem before expressing external disease symptoms. Interestingly, basipetal spread of the fungus as well as generation of internal and external necroses occurred more efficiently after inoculation of the excised apex than after mid-stem inoculation. In comparison, Úrbez-Torres et al. (2008) reported that L. theobromae caused larger basipetal than acropetal lesions in rooted cuttings of grapevine cv. Chardonnay and cv. Thompson, and on green shoots of grapevine cvs. Chardonnay and Thompson. Reports of these two types of colonization were also addressed in other research studies on wood colonization by various plant pathogenic fungi, including L. theobromae (Úrbez-Torres et al, 2009 and Bautista-Cruz et al., 2019); L. crassispora, L. euphorbicola and L. pseudotheobromae (Dianda et al., 2023), Neofusicoccum luteum and N. parvum (Billones-Baaijens et al., 2013).
The differences between external and internal colonization addressed in this study have not been considered in other investigations yet. In most of the previous studies only internal colonization has been studied (Berraf-Tebbal et al., 2020; Biju, et al., 2021; Briste et al., 2022; Dianda et al., 2023; Dong et al., 2020; El-Ganainy et al., 2022; Gunamalai, et al., 2023; Hernandez et al., 2023). In avocado, we measured a sum of acropetal plus basipetal internal necrosis of 230 mm in only 28 days of incubation. In comparison, Úrbez-Torres et al. (2008) calculated a lesion length of 338 mm when inoculating rooted cuttings of grapevine cv. Chardonnay and 183.1 mm in cv. Red Globe, with an incubation time as long as of 140 days. The same authors obtained lower values in green shoots of grapevine cv. Red Globe and in rooted cuttings of this cultivar after an incubation time of 180 days (Úrbez-Torres et al., 2009). Li et al. (2013), report lesion lengths of 149.1 mm in grapevine cv. Chardonnay incubated for 720 days.
Rapid generation of significant external and internal necroses caused by L. theobromae observed after inoculation of the excised apex indicates that this method is not only suitable for quantitative determination of virulence of field isolates of this fungus, but could also be an excellent and time-saving alternative in testing virulence of isolates of other members of the Botryosphaeriaceae.