Morphological Character Analysis and Signal Cryptic Speciation in Lasiodiplodia theobromae on Cashew ( Anacardium occidentale L . )

Lasiodiplodia theobromae (Pat) Griffon & Maubl. is a pathogen causing inflorescence dieback disease of cashew in Nigeria and also a common pathogen with a wide host range in the tropics and sub-tropics. The character variations in this pathogen necessitate better understanding of it towards development of management strategies. Isolates identified as L. theobromae were cultured from inflorescence dieback disease of cashew across growing ecologies of Nigeria and studied base on morphological characters. Variability in mycelial texture and colour, conidia and septa sizes and pycnidia production were recorded in this study. The Principal Component Analysis (PCA) and WARD clustering analysis identified four well-supported traits within the isolate group. Isolates within each cluster was: 2, 2, 4 and 1 respectively and isolate CDA1416 (Obollo-Afor) and CDA2924 (Idi-Ayunre) in cluster III were the most similar. Members within clusters I and II united at the semi-partial R-Square distance of 0.0294 and 0.0278 respectively. Isolate CDA2308 (Oro) was distinguished among others and signal a potential cryptic specie, differences in these isolates were supported by conidial morphology and textural variations. This understanding will form the bases for development of diseases management strategy against the pathogen.


Pathogen of Disease on Cashew
The analysis of morphological characters of Lasiodiplodia theobromae (Pat) Griffon & Maubl.was carried out as part of general study on character variations in this pathogen.Lasiodiplodia theobromae is an important plant pathogen in the tropics and subtropics.

Status of Disease in Nigeria
This pathogen has been reported to cause numerous diseases, including dieback, fruit rots, leaf spot among others (Punithalingam, 1980).Inflorescence dieback of cashew was reported by Adeniyi et al. (2011) and is a major limiting factor affecting cashew nut production in Nigeria, causing annual crop loss of 40-45% (Olunloyo, 1983).This disease is a major focus of recent cashew pathology research in Nigeria.In-view of the wide spread of this pathogen, the large number of hosts and its character variability (Adeniyi et al., 2016), it is possible that L. theobromae on cashew composed of a number of traits considering diversity in their morphological characters expression.The purpose of this study was to determine variability within a collection of isolates previously identified as L. theobromae through analysis of their morphological quantitative parameters as part of measures to develop management strategies against the pathogen.

Methods
A collection of isolates previously identified as L. theobromae, pathogen of inflorescence dieback disease of cashew in Nigeria (Adeniyi et al., 2011) was studied (Table 1), to investigate its mycelia and morphological characters according to the methods of Adeniyi et al. (2016).Such characters include mycelia growth rate, colour pigmentation, pycnidial production and conidial dimension of the isolates.

Analyses Selected Parameters
The data of the quantitative parameters were subjected to analysis of variance, using PROC GLM in SAS.Means of the measured parameters were separated using the honest significant difference method of (Tukey, 1949).Variability among the isolates for the colony colour and texture at the obverse and reverse were nominally coded.Mean of the two quantitative traits were generated for each isolate.The quantitative means across the three replication and the nominal codes for each isolate for the four parameters gave a 9 × 6 multivariate data matrix.The data was standardized (mean = 0; standard deviation = 1) to harmonize the various units of measurements among the parameters following the approach of Adewale et al. (2013).The data matrix was then subjected to Gower genetic distance (Gower, 1971) analysis.The resulting product was further subjected to Principal Component Analysis (PCA) and WARD clustering analysis.Mean and standard deviation was estimated for the members of each of the four clusters identified at the 0.1 inflection point on the dendrogram to be able to understand variability within each cluster.

Results
The sources of the nine isolates of L. theobromae from inflorescence dieback disease of cashew were cultured from major cashew growing ecologies of Nigeria were presented in Table 1.

Sampling Location
The randomly selected farmers' fields in major cashew growing ecologies of Nigeria were established over a decade, the agronomic observation perepectives, characteristics and assessement of the cashew plots showed closed and interlocking cashew canopies and irregular spacing of the plantations.The farmers' fields also comprised of cashew stands of varied sources and accessions mixed on same plot and the plots lack proper and expected farm management practises.

Description of the Pathogen and Isolates
The paraphyses of L. theobromae showed dark flask-shaped.The hollow structure of mature pycnidia was elongated to form a neck having circular pore through which conidia were liberated.The immature conidia observed in this study were globose and light brown but becoming dark-brown with longitudinal striation and single septation when matured.
With the four morphological traits employed in the study, the first three principal component axes captured 91.4% of the total variation (Table 4).Eigenvalues and variance proportion consistently decreased from PC1 to PC4.The proportional contribution of each character to the total variance (eigenvector) varied in dimension and quantity in the four PC axes.Most positive contribution in each axis was much higher than 0.3 and each character differently made highest contribution to the variance in each axis (Table 4). jas.ccsenet.5).

Discussion
The inflorescence dieback disease affect cashew at varied stages of flowering and different cashew accession are susceptible to this disease in Nigeria (Adeniyi et al., 2017) The paraphyses of L. theobromae, the dark flask-shaped and hollow structure mature pycnidia in this study is in agreement with the description of (Ekundayo & Haskin, 1969a, 1969b) as the ostiole was elongated to form a neck having circular pore through which conidia were liberated.The natures of conidia of L. theobromae observed in this study are similar to the descriptions of Meredith (1961) for the structure and variation in conidia sizes of L. theobromae.Alves et al. (2008) also reported presence of paraphyses within the conidiomata and the conidia initially hyaline and aseptate but with time a formation of septum and dark brown and melanin deposition gave conidia a striated appearance.Adeniyi et al. (2016) reported significant variations observed in characters of L. theobromae isolates from dieback diseases of cashew and the similarity was observed in this study on conidia size (31.3 -42.9 × 15.6 -19.5 µm) of CDA1416 and conidia size of L. gonubiensis reported by Pavlic et al. (2004) except for the multi-septate conidia nature of L. gonubiensis.However, conidia sizes of isolates in this study were somewhat larger than those reported by Burgess et al. (2006) and Alves et al. (2008) for L. theobromae.The diversity and cryptic speciation in the isolates of L. theobromae can be further established using molecular characterization tools.Wider isolate collection areas/locations will substantiate the level of biodiversity of this pathogen on cashew agro-ecologies of Nigeria and effect of good agricultural practices could also be evaluated.

Conclusion
The differential characters in the isolates of L. theobromae were evident in the conidial morphology and textural variations.This understanding will form the bases for development integrated crop and diseases management strategy against the pathogen to enhance yield of cashew.
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Table 1 .
Baseline information on isolate sources Note. *CDA: Culture of Dele Adeniyi.NC: North Central.SE: South Eastern.SW: South Western.

Table 5 .
Intra-cluster variability of the nine isolates based on two quantitative and two qualitative morphological characters